Target specific screens and their use for discovering small organic molecular pharmacophores

ABSTRACT

The invention relates to a general process by which recombinantly derived variable domains of antibodies encompassing either or both light and heavy variable regions with or without respective constant regions are engineered and selected for identification of unique surface domains of pharmaceutical targets or parts thereof which regulate target function. The recombinant antibodies are useful as reagents for high volume, rapid screening of occupation of the active surface domains by natural or synthetic entities. This invention is also directed to elucidating the three dimensional conformation of the antibodies, or parts thereof, which bind to the pharmaceutical targets and confers activity. Methods for creating high resolution molecular models which can direct the synthesis of biologically active small organic molecules useful as viable discovery drug leads are also provided.

This is a continuation-in-part of abandoned U.S. application Ser. No.08/286,084 filed Aug. 3, 1994 and which is incorporated in its entiretyherein.

FIELD OF THE INVENTION

The invention relates to a general process by which recombinantlyderived antibodies (rVab) are engineered and selected to identify uniqueactive surfaces of pharmaceutical targets. These recombinant antibodiesare useful as reagents to identify natural or synthetic entities whichoccupy active surfaces of pharmaceutical targets and which therefore maybe useful as therapeutics. This invention also relates to elucidatingthe three dimensional conformations of the various rVabs which bind tothe pharmaceutical target and confers target regulation and the use ofhigh resolution molecular models to identify or synthesize biologicallyactive small organic molecules useful as viable discovery drug leads.

BACKGROUND OF THE INVENTION

Today there are many approaches to identifying chemical entities whichhave a desired effect on a pharmaceutical target and therefore potentialas drugs. Common to all of these processes is the sequential use ofmultiple assays to identify a test compound's composite activityprofile. This activity profile usually consists of information on fourbasic attributes: potency, activity, selectivity and specificity.Selectivity indicates the ability to distinguish among closely relatedmembers of a particular target family. Specificity is the ability todistinguish between unrelated targets. Only two types of assays are usedto develop the activity profile of a potential drug: one, a bindingassay to measure affinity (i.e. potency) of the compound; and a second,an activity assay, to measure the compounds effect (i.e. agonistic orantagonistic) on the target. Binding assays measure the formation of thecomplex between target (T) and ligand (L). Targets include receptors,enzymes or structural components. Ligands include signals such ashormones, neurotransmitters, growth factors or test compounds. Untilrecently, L was labelled in some fashion (L*) for identification andquantitation of the L:T complex. Recently, binding assays have beendeveloped which use a tagged R (R*) to assess L affinity (see below).All these processes of labelling and R:L complex isolation andquantitation are known to those skilled in the art and have beenreviewed.

In the process of searching for small organic molecules with appropriatepotency, activity, selectivity and specificity for a particularpharmaceutical target, the order of testing is most often affinity,activity, selectivity and then specificity. In addition, some form ofbinding and/or activity assay, is interspersed with synthetic chemistryefforts at improving the compounds attributes. This generates aniterative cyclic discovery processes in which various assays andsynthesis are repeated over until a compound possessing all of thedesirable properties is obtained.

The present iterative process, although successful, is extremely timeconsuming and has a high probability of failure for several reasons.Although binding and activity assays have now been automated, screeningtakes significant time as it is done on individual entities withinchemical files containing over 100,000 entities. In addition, theproperties of potency, activity, specificity and selectivity areseparable, such that the presence in a compound of any one property isnot predictive of attaining another. For example, binding assays give noconclusive data on the activity (i.e., a compound with high affinity maybe an antagonist), and activity assays do not predict selectivity oraffinity. As a result, modifying a compound so as to change one of itsattributes (i.e., agonist activity) without modifying another (i.e.,target affinity or selectivity) is unpredictable and considerable timeis added to the discovery program when high affinity compoundsidentified early in the discovery process turn out to have inappropriateactivity or selectivity.

The relatively large number of biologically active small organic ligandshaving different general structures and which are capable of binding toa particular pharmaceutical target suggest that the binding surface ofthe target is not singularly unique. Furthermore, binding assays usingan endogenous ligand or close analog thereof are inherently biased tocompounds which bind to only a fraction of the available surface of thetarget. Even where the labelled ligand is not an endogenous one, thisconfinement means that the vast majority of active compounds identifiedby this process will be greatly restricted to the surface domain of thetarget which is used for interaction with the endogenous ligand.

This limitation is often viewed as desirable because the recognitiondomain for the endogenous ligand are those known via previous studies tohave the ability to modify target activity. However, investigation ofonly one target area severely restricts the ability to identify usefulligands. As endogenous ligands in most instances are agonists peptidesas in the case of opiate receptors, antagonist discovery can become arare event. In addition, because endogenous binding domains oftenexhibit limited diversity among receptor members of a single targetfamily, it becomes difficult for active compounds to discriminate amongtarget family members. This often occurs when the endogenous signal forthe family is a single entity and not a group of closely relatedentities. Acetylcholine (ACh) receptors are an example of a targetfamily with only one signal entity. The catecholamine receptors are anexample of a target family with a few but highly related endogenouscatechol signals.

In many cases, target diversity is found in target domains other thanthe specific binding site of the endogenous ligand. Some of thesedomains may be associated with the target's other functions, i.e.,signal transmission while others are quiescent domains not being used byany endogenous signals recognition or transmission. An example of adilemma in discriminating among target family members is that found forthe muscarinic receptor family (AChRm) where the binding domain foracetylcholine is used to monitor a test compound's potency, yet findingAChRm agonists which distinguish among the five ACHRm subtypes hasproven illusive to date.

The task for drug discovery is to devise a screening approach whichprovides detectable ligands to be used to screen compounds which bind tothe target and provide information regarding potency, activity,specificity and selectivity, as well as the three dimension (3D)conformation of compounds active at that particular site on thepharmaceutical target.

As part of any solution of these problems it is also necessary toestablish binding assays which report the interaction of test compoundswith allosteric modulatory sites on targets. An allosteric site is onewhich modifies the endogenous ligand binding site yet is discontinuousand non-overlapping with that site. Such target sites have importantphysiological and pharmaceutical consequences and have been reported.For example, the allosteric site on the Gaba A receptor bindsbenzodiazepines (BDZ) and thereby modulates the binding of theendogenous neurotransmitter Gaba. Occupation of the allosteric BDZ site,which can be done by chemicals from many unrelated structural groups,has a significant and recognized therapeutic influence on physiologicalprocesses including anxiety and sedation.

It is also known that active allosteric sites exist which are modulatoryfor endogenous ligand binding and have observable effects of their ownon the target. Such an allosteric site is present on the Gaba receptor.[Garrett, Blume and Abel 1986; Garrett, Abel and Blume 1986].

Present screening techniques which monitor direct binding of testcompounds to allosteric target sites are not routinely done because a)high affinity tagged ligands which bind to these sites are usuallyunavailable at the start of a discovery program; and b) the necessarymonitoring of detectable endogenous ligand dissociation or bioassays aretoo time consuming in initial screening protocols. Without a simple,rapid and comprehensive way to observe all potential target sites,investigation of the surface of a pharmaceutical target for potentialmodulation remains limited to a small part of the target surface. Newmethods are necessary to survey the entire target surface in earlyscreening for discovery leads.

Recently methods of identifying various entities which recognize targetsurfaces have been reported which do not depend upon the availability oftagged ligands with high affinity for the target. [Delvin, J. J.,Panganiban, L. C., and Devlin, P. E., 1990]. These assays detect acompounds surface recognition activity directly via formation of anidentifiable tagged target (T*):Ligand complex. In one version, testcompound is coupled in identifiable compartments to a solid matrix ofvaried composition at concentrations which allow sufficient amounts oflabelled target to bind and form stable ligand-labelled target complexesfor subsequent detection via chemical, radioactive, or biologicalmethods known to those skilled in the art. Subsequent isolation (oridentification) of test compound from the compartments containinglabelled target provide active chemical structures. In one such versionwhere test compounds are free oligonucleotides, the oligonucleotides areisolated in complexes with the target, and are amplified and sequencedby PCR technology. [Delvin, J. J., Panganiban, L. C., and Devlin, P. E.,1990].

Phage display is a particularly sensitive method of presenting peptidetest compounds to a target. Phage may be engineered to express the geneencoding the test peptide as a fusion protein with one of its surfaceproteins. Methods involving phage display are referred to in Winter etal. PCT application WO 92/20791; Huse, WO92/06204; and Ladner et al.WO90/02809.

Although these newer approaches have now been incorporated into randomdrug screening protocols, they do not resolve the following problems:the assays of the critical attributes of potency, activity, selectivityand specificity are still unconnected; active target surfaces includingthe endogenous ligand site and allosteric sites have not beenidentified; and 3D information on conformation of the active agent isnot provided. More importantly, most of the agents available forscreening, i.e., peptides, nucleotides, lipids, and carbohydrates whichare available in large libraries, are not totally satisfying asdiscovery leads because none are expected to be orally active, or passmembrane barriers to get at intracellular or central nervous systemtargets. In addition, these classes of compounds are so flexible as toobscure their active 3D-configuration to such a degree as to prevent orseverely limit their use as models for organic synthetic efforts. Animprovement in screening would then encompass a resolution of thesedeficiencies so that these broad surface recognition libraries couldattain their full usefulness.

In covering the prior art for high throughput binding screens for targetmodifiers, it also is necessary to review what is known of theendogenous ligand signals as well as their targets. Both shedsignificant light on additional problems and limitations encountered inthe binding assays available today for discovery approaches.

Endogenous ligand signals are those ligands which directly modify targetactivity. The size of endogenous ligands varies greatly, ranging from100 Daltons (e.g., as for glycine in its regulatory role as anexcitatory amino acid neurotransmitter) to over 100 kD (e.g., as forsome extracellularly active growth factors (GF) with a proportionedincrease on surface area. The composition of endogenous ligand isequally varied including organics such as neurotransmitters; peptidese.g. somatostatin, LH, LHRH and TRH; proteins eg., growth factors; andlipids; carbohydrates; and inorganics such as ions.

For discovery purposes, common to all is the desire to replace theendogenous ligand with a small organic molecule. The problem ofscreening for replacements appears to be very different for most smallendogenous ligands, i.e, neurotransmitters and neuropeptide modulatorscompared to large endogenous ligands i.e., hormones, growth anddifferentiation factors. Although small organic molecules have beenfound which can be active at targets for small endogenous ligands, few,if any have been found for the larger molecules such as proteins.

Corresponding to the diversity in endogenous ligands is the equallyextensive diversity in target domains which are responsible forrecognizing (i.e., binding) and responding to endogenous ligand signals.It is generally accepted that both signal and target have specificdomains involved in forming the actual contact points found within theendogenous ligand:target complex (EnL:T). Recent data on crystallizedgrowth hormone (GH) and its receptor complex provides detailed molecularinformation on the amino acids within the GH hormone ligand and itstarget GH receptor interactive domains.

Recent data on the crystal structure of GH and its receptor has shown asingle GH molecule to contact the same set of amino acids in each of twoidentical GH receptor units complexed with one GH molecule. [Cunninghamand Wells 1989; Cunningham et al. 1991; DeVos, et al. 1992]. Each of thereceptor units therefore has only one target site which is the same onboth units. Each receptor uses the same 7 amino acids to define thebinding site which participate in GH binding and receptor dimerizationnecessary for activity. [Cunningham and Wells 1989; Cunningham et al.1991; DeVos, et al. 1992].

Dimerization of at least two receptor subunits by monomeric ormultimeric hormones is required for receptor activation for the majorityof hormones studied to date, such as growth factors, including nervegrowth factor (NGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), interleukins (IL2, 4 and 6), interferons and insulin.[DeFronzo, Bonadonna, and Ferrannini, 1992; Bamborough, Hedgecock andRichards 1994; Kishimoto, et al. 1994; Claesson-Welsh, 1995]. In somecases, the two units of the hormone, as well as receptor are notgenetically related. In such cases one subunit provides high affinityhormone binding and the other intracellular signalling (e.g., tyrosinekinase activity). [Ullrich, et al., 1986; Kaplan, Martin-Zanco, andPatrada, 1991; Kaplan, et al. 1991; Klein, et al. 1991; Argetsinger, etal. 1993; Obermeier, et al. 1993; Weiss 1993]. In some cases, the loweraffinity receptor when dimerized can be activated. [Ullrich andSchlessinger 1990; Stahl and Yancopoulos 1993; Claesson-Welsh 1995].

Among many hormones and hormone receptors, it is now apparent that anunexpected and unanticipated degree of structural homology exists withsubgroups of these signals and receptors forming homologous familieswhich sometimes follow along different genetic evolutionary lines. Otherfunctional similarities may be brought about as a result of convergentevolution. In either case, the active 3D conformations of ligands andreceptors appear to follow some general principals. However, for drugdiscovery, the principals gleaned from these studies have not yet beendetailed enough to bypass crystallography of particular hormone/receptorcomplexes in order to gain sufficient specific information as to deducethe molecular shape of active small organic molecules.

Deciphering the elements necessary in a signal to activate ahormone/growth factor receptor has included (1) crystal formation andanalysis at <3 Å of receptor and endogenous ligand complexes; (2) theinfluence on function (i.e, ligand binding and receptor activation)caused by molecular biological mutagenesis of single amino acids orshort peptide deletion/replacement, or chimera formation of both thehormone and receptor units. In addition, monoclonal antibody binding tosurface domains available when ligand and receptor are eitheruncomplexed or in the R:L complex, along with the ability of Fab2 versusFab1 to activate or block receptor activation in vitro, in situ or invivo has been studied.

The above studies when taken together, provide information concerning(1) the contact points between hormone and receptor; (2) the amount ofenergy of binding involved in these contact points; (3) amino acidsoutside of the receptor:ligand contact points essential for globalreceptor/ligand stability or dimer stability, or receptor signallingactivity (i.e. tyrosine kinase, binding of other intracellularregulatory factors, internalization, uncoupling for effector system).

Critical for identifying small organic molecules which are active athormone receptors are the data from the above indicating (1) number ofunits/active complex; (2) amino acids of the target specificallyinvolved in the binding domain with the endogenous ligand; and 3) aminoacids of the ligand specifically involved in binding and/or activatingthe target. Of all of the above information, clearly the rate limitingevent today is obtaining sufficiently resolved crystallography data ofhormone/receptor complexes. However, complexes of receptor and ligandare often difficult to identify and crystalize thus preventing one fromobtaining the structural information. It is also recognized that thevarious molecular, biological, immunological studies, biochemical andpharmacological studies noted above, also take considerable time andeffort. Accordingly, prior art approaches to identifying active smallorganic molecules are long and arduous with unpredictable results.

In the approach outlined above, it is important that both structural andbiological data be obtained as each has its own limitations andartifacts. Also, contact points could reflect specific aspects ofcrystal formation which do not reflect the structure at the protein insitu, or the crystal may contain an inappropriate number of subunits. Onthe other hand, the biological data generates both false positives andnegatives. Furthermore, if antibodies are used to probe the binding siteof the target, not all receptor or ligand surfaces may be immunogeneticaccessible to Fab2 or Fab1 antibody. Another problem is the difficultystudying allosteric sites which do not interact directly with the signalligand.

Despite considerable effort, a major problem in drug discovery has beenthe identification of small organic molecules capable of activatingpeptide hormone/growth factor receptors. This is likely the result ofthe multivalent nature of endogenous ligands for these receptors and therequirement to dimerize or simultaneously activate multiple attachmentsites on a single receptor (receptor subunits) for receptor activation.Even for receptors which are homodimers, such as GH receptor (GHR), asingle small organic molecular monovalent attachment to the GHR site Iis not sufficient to cause activation, nor displacement of growthhormone from its active divalent dimer receptor complex.

Failure to find single small organic molecules in conventional bindingassays stems from the fact that the labelled hormone is bivalent, andits displacement from two receptor units by a single monovalent smallorganic molecule (i.e. compounds which attach to only one receptortarget at a time) is thermodynamically unfavorable in the present daybinding assay. Furthermore, in the large majority of cases the receptorfor a given hormone is a heterodimer. Thus, for a given hormone/growthfactor-receptor binding pair, there may exist at least two differentbinding sites on the target which may be due to the multimeric nature ofthe target or a target consisting of allosteric sites on a monomericunit. In all of these cases, the endogenous ligand must thereforecomprise at least a sufficient number of binding sites which areproperly spaced to bind to the multiple sites on the target necessaryfor activation. Obviously, one would then require a multimeric or amultivalent small organic molecule for displacement of these hormonesfrom their targets.

Given the complexity required of each small organic molecule to bind thereceptor at the multiple sites necessary for activity, or to displacethe endogenous ligand, one could expect that the occurrence of a singlesmall organic molecule with two unrelated yet active binding domainswould be equal to the chance of finding one multiplied by the chance offinding the other independently. As active small organic molecules arefound by random robotic assays at a frequency of between 1/1000 to1/10,000 on most screens for ligands requiring only one binding site onthe ligand, and which have correspondingly a single binding site on thereceptor, one would expect to screen an organic chemical librariescontaining from 10⁶ to 10⁸ compounds in order to identify an activemolecule. Such libraries exceed those which could be screened in somereasonable assay format and actually exceed most made by even thelargest pharmaceutical companies.

Therefore, a different approach to screening for small organic moleculeswhich can activate hormone receptors is needed.

A number of libraries now exist for screening such large numbers. Twohave been noted already, the oligonucleotide and peptide library.Another such file contains natural products.

Classical chemical libraries consisting of synthetically derived smallorganic molecules are routinely available from commercial sources (e.g.Alldrich, and Kodak) and consist of upwards of a 1-200,000 entities.Recently a survey of the chemical entities within such librariesuncovered 100,000 or so chemical structures as being the cores uponwhich most of the individual entities were crafted. The averagemolecular weight of the entity within such files ranges between 200-400Daltons which would account for no more than one such contact site pertarget.

Screening of small chemical compound libraries is limited only by theiravailability, which most often is <100,000.

With the advent of molecular biology and gene cloning and sequencing, ithas been discovered that most pharmaceutical targets are not uniqueentities unto themselves, but in fact belong to families of sometimesrather large size and close relatedness. Recognition of this fact hasmandated a much more serious look at all of the members of the family towhich the target under investigation belongs so as to identify leadcompounds which can distinguish among its family members. If one usedonly binding assays as a primary screen for potency, activity,selectivity and specificity, one would require affinity labelledstandards for each of the family members. Although this is potentiallypossible when the endogenous ligand signal are proteins due to theirnative affinity and ease of labelling, it is not presently feasiblewhere small organics are the only known signals. This approach is alsounsuitable for targets with unidentified signal ligands. Any discoveryof how to include such widespread specificity testing into primarybinding screen assays would greatly increase the probability of drugdiscovery success.

SUMMARY OF THE INVENTION

This invention provides compositions and methods for identifying activesurfaces of biologically active sites of pharmaceutical targets.Identification of these sites is useful for preparing reagents suitablefor use in screening assays of small organic molecules to identify thoseas candidate lead compounds possessing desired attributes of biologicalactivity, specificity, selectivity and affinity.

Reagents are provided by this invention which are suitable foridentifying active sites on pharmaceutical targets. The reagentscomprise libraries of variable regions of antibodies obtained andmodified by molecular biology techniques which are used to preparerecombinant Fab fragments (rVab) useful for scanning the surface of atarget in a manner so as to identify those rVab's having desiredpotency, activity, specificity and selectivity. The attributes ofpotency, activity, specificity and selectivity are collectively referredto as a "composite activity profile" (CAP). The rVab's which are madeand identified by this invention as possessing the desired CAPattributes specifically bind the target (i.e. are T⁺), are selective forthe target (S⁺) and activate the target or are capable of activating thetarget when combined with another ligand (A⁺).

By combining structural features of various members of the recombinantantibody library which possess activity at a defined pharmaceuticaltarget, this invention provides a method of determining a compositestructure possessing the desired composite activity profile. Thiscomposite structure may then be used to identify small organic moleculescapable of acting at the target surface with either agonist orantagonist activity with the sufficient specificity and electivity.

The method according to this invention of identifying ligands capable ofbinding to active sites and possessing a composite activity profile fora given pharmaceutical target comprises combining members of arecombinant antibody library with a pharmaceutical target coupled to areporter which reporter is capable of signaling activation or inhibitionof the pharmaceutical target. Reporters of pharmaceutical activity mayinclude but are not limited to, for example, receptor coupling tomodulators such as the G protein; oligomerization of receptor subunits;changes in enzymatic activity such as kinase activity; or changes in ionflux. According to this method, individual members of the librarypossessing desired activity as demonstrated by the reporter, are usefulindividually or collectively in subsequent assays to identify smallorganic molecules capable of possessing the desired activity at thepharmaceutical target. By combining structural features in commonbetween multiple members of the library possessing the desired activity,a composite structure for activity may be derived which may then be usedto create a model for a compound possessing the desired activityattributes.

This invention also provides a method of identifying small organicmolecules which are active at the target sites comprising screeningpotential drug candidates in a binding assay for their ability todisplace labelled, rVab members possessing a desired composite activityprofile consisting of potency activity, selectivity and specificity forthe pharmaceutical target.

Small organic molecules as candidates for drugs may also be identifiedby analyzing the structure of the model derived from the structure of atleast two active members of the rVab library and determining commoncharacteristics including, but not limited to charge and spacialorientations which participate in binding to the active sites of thepharmaceutical target. Using the model, small organic molecules may beobtained by synthesizing compounds possessing the common structuralfeatures identified in the model, or screening a chemical file data basefor members possessing features in common with the model.

This invention also provides means of identifying structuralrequirements of ligands capable of binding to pharmacological targetscomprising multiple binding sites existing on one or more molecularentities which when bound by a single ligand are capable of activatingthe pharmacological target. Similarly, this invention provides a meansof identifying structural requirements of multivalent ligands capable ofactivating pharmacological targets comprising binding sites too large tobe occupied by a monovalent small organic molecule or requiringconcurrent binding of a multivalent ligand to effect oligomerization ofseparate molecular entities to form an active pharmacological target.

This invention also provides reagents comprising recombinant antibodylibraries (rVab's) which have been constructed to encode CSR and CDRregions with specific variations and in which the CDR and CSR regionsare expressed on a specific identifiable framework structures.

The recombinant libraries of the invention may be packaged in variousforms including bacterial phage which express the recombinant antibodieson their surface.

It is therefore an object or the present invention to provide a processfor the identification of small organic molecular replacements capableof modifying a pharmaceutical target with a desired composite activityprofile comprising sufficient potency, activity, specificity andselectivity to be considered as an initial drug discovery lead.

It is a particular object of this process to identify surfaces of apharmaceutical targets capable of discriminating among members of afamily of related targets which are activated by the same or similarendogenous ligand or utilize similar signal transduction mechanisms.

It is a particular object of this process to identify active orregulatory surfaces of a pharmaceutical target which may or may not beused by an endogenous ligand for the target of interest, and which isnevertheless capable of modifying the pharmaceutical target in somepharmaceutically useful manner.

It is a particular object of this process to identify allosteric siteson the pharmaceutical target which are not used by endogenous signalsnor have activity on their own, as well as active allosteric sites whichare used by endogenous signals other than the pharmaceutical targetactivating signal and which have some type of activity on their own.

It is a particular object of this process to provide a repertoire ofsurface recognition libraries which together recognize diversepharmaceutical target surfaces by constructing a small number ofcombinatorial antibody libraries.

It is a particular object of this process to convert by a single simpleand rapid process any unlabelled recombinant variable antibody fragment(rVab) isolated from a library to a labelled one to act as a reagentcapable of identifying small organic molecules which possess any one, orcombination thereof, of the attributes of potency, activity, specificityor selectivity simultaneously when screening random chemical libraries.

It is an object of this process to identify the specific binding regionsof pharmaceutical targets requiring binding to sites in at least twodifferent regions to cause a response of the target. Such regions may bepresent on monomeric or oligomeric pharmaceutical targets. Theendogenous ligands for such sites generally are multivalent monomeric oroligomeric proteins which bind to the multiple regions which define theactive surface of the pharmaceutical target.

This invention provides a method for identifying the structuralrequirements for ligands to bind at the separate regions and identifyingsuch ligands. By combining the ligands capable of individually bindingto the separate regions into a single molecule, fully active ligands areprovided.

It is another object of this invention to identify the monovalentdeterminants making up the active surfaces on the targets for largeprotein signals such as hormones and growth and differentiation factorsconsisting of oligomeric receptors. Such receptors may containhomologous or heterologous components with one or more of these unitsobtaining a part of the signal recognition determinant. It is aparticular object of this process to use chemical oligomerization ofsmall organic molecules for each of multiple binding sites to derive anactive oligomer for large proteins such as growth factors and hormoneswhich contain multiple binding sites within their active bindingdomains.

Accordingly, another object of this invention is to identify smallorganic molecule replacements for large protein signals such as growthfactors and protein hormones be they allosteric or competitive modifiersand whether they be monovalent or multivalent.

It is a particular object of this invention to identify small organicmolecule replacements for pharmaceutical targets which have nobioorganic endogenous ligand signals, such ascertain ion channels,pumps, and exchangers.

It is a particular object of this invention to provide high volumebinding assays which discriminate agonist from antagonist small organicmolecule replacements.

It is a particular object of this invention to be able to identity fromlarge antibody variable region libraries, individual variable regionswhich distinguish from one another binding sites which conferselectivity of pharmaceutical targets for specific members of a genefamily.

It is a particular object of this process to provide labelled antibodyvariable regions which interact with and modify the activity of targetswhich have no identified endogenous ligand, nor exogenous naturalsignals, and which labelled ligands have sufficient affinity for thepharmaceutical target to be used in competing binding assays in whichsmall organic molecules may compete for binding with the labelledligands.

It is another object of this invention to provide a plurality ofdifferent recombinant antibody variable regions which recognize at leastone common binding site of a pharmaceutical target and whichcollectively provide structural information useful for designing activesmall organic molecules which are active at the pharmaceutical target.

It is another object of this process to provide a general method torapidly obtain peptide structures which are useful as 3D modelscomprising the minimum characteristics of small organic moleculereplacements which have sufficient potency, activity, selectivity andspecificity to classify as viable discovery leads.

It is a particular object of this process to provide molecular modelsfor active ligands wherein the pharmaceutical target necessary to beoccupied by active ligand comprises one or more binding sites on one ormore molecular entities.

It is a particular object of this process to be able to solve thecanonical structures of the CDR VH3 of recombinant antibodies which havebeen identified as possessing the desired properties of potency,activity, selectivity and specificity.

It is a particular object of this process to be able to use compositestructural characteristics to direct a synthetic effort capable ofdirectly synthesizing active small organic molecules.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Stages of the Topographic System Assay (TSA). FIG. 1 shows theactivities and products of the three main stages of the TSA. Whencombined together, Stage I and II, or Stage I and III, allow theidentification of small organic molecules (SOMERS) which are active atpharmacological targets (T). A MULTIMER is at least two SOMERscovalently linked together to produce an active molecule. A BEEP is abiologically enhanced ensembled pharmacophore, and Tn is subunit n ofpharmacological target.

FIG. 2. Related Antibody Structures and Variable Region Domains. A.Shows various forms of antibody structures including the variable (V)and constant (C) regions of immunoglobulin (Ig) heavy (H) and light(L)chains. Antibodies constructed in this invention by molecular biologytechnology have a r prefix. B. Shows details of the antigen recognitionVariable region (V) domains of the VL and VH. FW is the `constant`framework regions; "CDR" refers to the complementary determining regionsas defined by Kabat (Kabat 1991); CSR refers to canonical structuresfound in CDRs as originally defined by Chothia (Chothia and Lesk, 1987);V (with leader sequence), D (diversity) and J (V/C junction) are thegenes which are combined to create the mature VH and VL genes. V Regionsare attached via genetic recombination for VL to either a kappa orlambda Constant region. VH are recombined with three Constant regions insequence with CH1 being attached to VH. The V regions of the inventioncan used either without C regions, or with kappa or lambda for CL, andup to three C regions for CH.

FIG. 3. Potential Planar, Cavity and Grove Antibodies of KnownCrystalline Structure for rVab Library Construction. FIG. 3 lists anumber of antibodies for which there data is known concerning theircrystalline structure and which are potential parental antibodystructures for construction of the rVab library as described in thisinvention. The antibodies are grouped according to their type ofantibody combining site: i.e., planar, grove or cavity-type structure.

FIGS. 4A and B. Comparison of Natural Fab and rVab LibraryDiversification. A Nature's Immune Repertoire. V, D and J are the genesrecombined to make the mature V gene; rf* are the reading frames of theD gene which can be used to make sense protein sequences uponrecombination with V and J. CDR* are there are no CSRs for the VH3region. The number of known CSR for each CDR is given in parentheses. B.The rVab Repertoire: Diversification arises by using all permutations ofthe known CSRs, 3 different length CDRH3 and randomization of aminoacids at two positions within each CSR (or CDRH3) within a single VH andVL parental framework structure. Primary randomizations are made duringconstruction of the rVH and rVL (see FIGS. 7, 8) and allows all 20essential amino acids to appear at given positions within V regionsamong members of the rVH and rVL libraries. CDRH3 are three known CRDH3sof different sequence and three different lengths covering VH amino acidpositions 95-102 (see text for details). rVab is encoded by one rVHCH1and one rVLCL gene on the same piece of DNA. Totals of CSR include CSRand CDRH3 combinations.

FIG. 5. Type and Diversification of Amino Acids at various positionswithin V region. Numbering of the amino acid (AA) positions as per Kabat(Kabat, 1991). Library Diversification identifies the high prioritycandidate amino acid position for primary library diversification duringconstruction of rVH.lib and rVL.lib as described in this invention.

FIG. 6. CDR and Canonical Structures (CSR) of V Regions. Particularamino acid (single letter code) at V gene positions critical forparticular CSRs are given as defined by Chothia (Chothia and Lesk,1987). * represents amino acids not within CSR or CDR which participatein defining the CSR. The diversity position is the amino acid positionused for primary library randomization as described in this invention.

FIG. 7. Construction of the rVLCL Lib. of Diversified Canonical CSRs:rVLCL.Lib. A-F are sequential steps of the process constructing rVL.Lib.G is the final step of recombination of rVL.Lib with a rCL to formrVLCL.Lib. Amino acid positions occur in brackets; nucleotide positionsare given left to right as 5'-3' in parenthesis; restriction sites (rs)also appear in brackets and have a "p" prefix to denote when they arelocated within the plasmid and not the V region. Restriction sites aredenoted by combinations of letters and numbers. Primer direction isdenoted by arrows (left is forward (FWD)), and right is a reverse (BCK)primer). * denotes more than one amino acid at a CSR position which iscritical for a particular CSR; Δ denotes that diversification byrandomization of amino acids with CSR or CDR has occurred. Lib suffixesindicate a library of many individual members. Heavy line indicates thatthe product(single entity or library) has been cloned in to plasmidpCLONALL (pC).

FIG. 8. Construction of the CSR and CDRH3 Diversified rVHCH1.Lib.Construction of CSRH1 and H2 and three CDRH3 of different lengths (i.e.,5, 7, or 10 amino acid insertions); diversification by amino acidrandomization and combination of CSR and CDRH3 in all possiblepermutations is as illustrated in a manner analogous to that describedfor rVLCL.Lib (see legend to FIG. 7).

FIG. 9. General Usage Plasmids. A. Illustrates the sequence ofrestriction sites (rs) which occurs in the cloning site of pCLONALL. Useof each in rVH.Lib and rVL.Lib construction is noted wherein "- - - "denotes a restriction site used and defined by parental AB sequence;wherein X denotes a restriction site not used in that particular rV.libconstruction. General positions of restriction sites within the rV andrC regions under construction are shown. JCH and JJCL are the naturalJ/C gene recombination region with included amino acid positions givenin brackets. JCLINK is the position of the J/C recombination restrictionsite, also referred to as rs3. B. Events used in constructing theplasmids carrying rC regions and in the final step of rVab.Libconstruction wherein rV regions are appended to rC regions. The twoplasmids needed for this are listed as pVxACCEPTORs. C. Plasmids used increating expression vectors for the rVHCH1 and rVLCL chains of the rVabwhen not attached to phage coat protein gpIII. EK is the enterokinasecleavage site. ISOTAG is the additional amino acid sequence useful inisolation and labelling rVab as rVab-REPORTER constructs.

FIG. 10. General Primer Table. Primers are written as 5'-3'. Numbers andsingle letters designate individual amino acid positions which in theprimers would be corresponding triplet codon sequences for the aminoacid at these positions. The letter N within parenthesis denotes therandom appearance of the nucleotides A,T,U,C used to randomize the aminoacid at this position. Letters, without parenthesis are used forsequences necessary for a desired CSR or CDRH3 structure; numbers areused for sequences which are not critical to CSR or CDRH3 structure. rsis a restriction site sequence. Sequences for all FWD primers arecomplementary to the sense sequence. Approximate primer sizes innucleotides are listed as #mer. The right hand column signifies generaluse of primer with amino acid randomization; and SEQ. is sequencing.

FIG. 11. Constructs for CRE-LOX recombinatorial formation of rVab.lib:PartI. Expression of rVab with or without one attached random octamerpeptide (Pep 8) library. FIG. 11 illustrates the steps generating thenecessary phagmid and plasmid constructs to allow in vivo recombinationof individual rVHCH1.lib and rVLCL.lib members, by the Cre recombinase,and the construction of a single phagmid containing an rVHCH1 and rVLCLmember on one piece of DNA (i.e., an rVab). This procedure is used forrVab.Lib construction where there is no need in the TSA discoveryprocess for subsequent addition to rVab of more than one random octamerpeptide (PEp8.Lib). Wild type (wt) and mutant (511) loxP sites are asdefined in legend to FIG. 12. LpelB and LgpIII are leader sequences forpelB and gpIII.

FIG. 12. CRE-LOX Plasmid and Phagmid Sequences used for rVab.libConstruction. For use in rVab.lib construction by in vivoCre-recombinase directed recombination of rVHCH1 and rVLCL onto singlephagmids where there is a subsequent need in the TSA process forattachment to rVab of no more than one random peptide library.

FIG. 13. Constructs for CRE-LOX Recombinatorial Formation of rVab.Lib:Part II. Expression of rVab with or without one or two attached randomoctamer (PEp8) peptide libraries. Steps involved in adding Pep8.Lib; i)illustrates expressing one peptide (PEP^(1at)) at the amino terminus ofVH (Pap8^(1at)); ii) illustrates expressing one peptide at thecarboxyterminus of CL (Pep^(1ct)); and iii) illustrates expressing onepeptide at the aminoterminus of VH (Pep^(1at) and one peptide at thecarboxyterminus of VL (Pep^(2ct)). Step E illustrates use of two primersrequired to append Pep8.Lib to either VH or CL.

FIG. 14. In vivo, Generation and Expression of rVab.Lib members. Thegeneration of rVL and rVH gene pairs (rVab) as one DNA molecule, as wellas the expression and phage display of rVab attached to coat proteins offd is illustrated. Synthesis of rVHCH1- and rVLCL proteins and theircomplexation to form gpIII attached rVab for phage display isillustrated showing cells, such as bacteria, infection of bacteria withphage carrying rVLCL and transformation with DNA plasmids carrying therVHCH1-construct; and in vivo recombination of rVHCH1 and rVLCL onto asingle fd via the LOX sequences and the P1 provided CRE-recombinase.Following recombination and replication, a combined single expressiblepair of rVab genes is packaged per phage. When induced, rVLCL is madeand introduced via its leader into the periplasmic space were itscomplexes under reduced conditions with synthesized rVHCH1-gpIII coatprotein to create the desired rVab complex attached to the gpIII phagecoat: protein (see text for details).

FIG. 15 Flow Diagram of Diversification and Simplification Paths of theTSA. Steps are outlined for optimizing TSA+ attributes of rVabs for agiven pharmacological target. The library attributes are potency ofbinding to Target (T), specificity and selectivity for Target (S) andregulation of target Activity (A). "+" denotes that the attribute ispresent in the rVab member.

FIG. 16. Isolation of Target (T+) Specific/Selective (S+) rVab. A.Isolation by panning for Target recognition(binding) (T+). B. Isolationby panning for Target Specificity and/or Selectivity (S+). Isolation ofT+ and S+ rVab can be done in any order, and when used together isolaterVabTS+ members. T denotes the pharmacological target; φ phage displayedrVab; com-T-pep represents the entity, holotarget, subunit or peptidefragment, which is to be distinguished from the Target. Binding to thecom-T-pep prevents rVabS binding to matrix attached T.

FIG. 17. Selection of rVab Scanners for Active Target Surfaces Used bySignals with Single Attachment Sites. FIG. 17 presents a ilow diagram ofthe TSA process isolating rVabTSA+ members from a rVab librarypreviously identified as T+S+. T, S and A are defined in legend to FIG.15. Native signal is the endogenous or previously identified agonistentity (e.g., protein, peptide, neurotransmitter) which activates Targetby interaction at a single attachment site (see Text for details).Allosteric Effector is an endogenous or previously identified entitywhich binds to a single attachment site on Target which modifies agonistactivity but has no activity on its own. rVabA+ are isolated bycompetition by native signal or allosteric change in T by allostericeffector which prevents normal rVabTS+ binding to T. The binding ofrVabTS+A-members is unaffected by the presence of the allostericeffector or native signal and therefore is not isolated free in thesupernatant during this process.

FIG. 18. Discovery of SOMERs for a Target with a Single Univalent ActiveSite. FIG. 18 illustrates the steps of the TSA process of rVab-Scannerto Reporter conversion and Reporter use in competitive binding assays toidentify active SOMERs for the pharmacological Target. Both competitiveand allosteric active SOMERs are identified in this process.

FIG. 19. Identification and Isolation of Active rVabTSA⁺ for theMuscarinic Acetylcholine Receptor subtype m1 (AChR_(m1)). (s)--denotesmatrix attached Wheat Germ Agglutinin; T, S and A are as defined in thelegend to FIG. 15; R denotes receptor target; G denotes guaninenucleotide binding protein; RG denotes RG noncovalent complex; φ denotesphage displaying rVab. The TSA process isolating rVab based onspecificity/selectivity (see FIG. 16) is illustrated for the isolationof AChR_(m1) rVabS_(m1) + using Agonist-Like rVabT+A+ (type 1 rVabTA+).The same process is used Nor isolating Partial-Agonist-Like,Allosteric-Agonist-Like and Competitive Antagonist-Like S+ rVabTA+(i.e., respectively type 2, 3 and 4 rVabTA+)

FIG. 20. Isolation of Active rVabTSA⁺ for Complex Active Sites onDimeric Receptor Targets (T₁₋₂). FIG. 20 illustrates the TSA process bywhich the rVab pair for each part of the active site on each of tworeceptor target subunits (T1 or T2) is isolated. The process is shown infull for one member of the pair; that for the active site region on T1,and is duplicated for the active site region on T2. m-T denotes matrixattached Target; comp-T-receptor denotes comp-T-pep as described in FIG.16. φ denotes phage displayed. Pep8.Lib is the random octapeptidelibrary displayed as a fusion protein with phage coat protein gpIII.Pep8T₂ + is the library of peptides which bind to T2. rVabT1-Pep8T2.Libis the rVabT1S+.Lib to which the PEp8T2+ Lib has been appended (seeFIGS. 12 and 13 for details of rVab-Pep.Lib construction). Preselectionof the T2+ Pep8.lib is not required and a random Pep8 Lib can be used inthis process. Testing for rVabS+ is optional and can be done at any stepalong the process. The related rVabT2m+S+A+-Pep8T1+ member of the activedomain pair is obtained in parallel analogous manner.

FIG. 21. Using Active Bivalent rVabT1-Pep8T2 to Screen for DisomerReplacements of a Multivalent Signal. FIG. 21 presents a flow diagram ofthe steps of the TSA in which each rVab member of the active pair ofrVab-Pep8 for both domains of the active site, which occur on separate Tsubunits are used to find a DISOMER replacement for the native signaland which regulates Target activity. [A+] denotes that the rVab-Pepentity is active in regulating the T1-2 dimeric Target. A* denotes thatthe rVabTS+ member is derived from a rVab-Pep entity which is [A+].DISOMERmn denotes covalent linked SOMERs for the pair of active sitedomains identified by the paired rVabTSA* members.

FIG. 22 Summary of the Discovery of DISOMERs for a Bivalent Hormone.

FIG. 23. Flow Chart of TSA Steps Creating and Using a BiologicallyEnhanced Ensembled Pharmacophore (BEEP).

FIG. 24. The TSA Process of Finding and Relating Sets of SurfaceAttributes of rVabTSA+ to Create a BEEP.

FIG. 25. The TSA Process of Finding the Surface Common to All ActiverVabTSA+ Scanners for an Active Site of a Target.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides methods and compositions for identifying ligandscapable of identifying active sites on pharmacological targets. Thisinvention utilizes recombinant antibodies which possess the combinedattributes of potency (affinity), selectivity, specificity, and activityas reagents useful for modelling active ligands and identifying smallorganic molecules which also possess these attributes and thereforeutility as drug leads or therapeutic compounds.

I. Pharmacological Tar gets Identified by this Invention.

Pharmacological targets may be receptors for endogenous or other ligandswhich evoke a physiological response by the cells on which the receptorsare present. Besides receptors, the pharmacological target may be ionchannels, transport proteins, adhesion proteins such as N-CAM, or anyother physiological regulatory surface which is excessible to beingidentified by the recombinant antibodies and which is activated by aspecific ligand. A non-limiting list of exemplary physiological ligandsfor which active surfaces may be identified by using the methods andcompositions of this invention are listed in Example 4.

Receptors may include those for neurotransmitters, hormones, growth ortrophic factors, modulatory peptides, ions or other moieties which actas signal ligands for the pharmacological target. Preferred nonlimitingexamples of neurotransmitter and peptide receptors for which activesurfaces may be identified include those for acetylcholine, i.e.,nicotinic, and the various forms of the muscarinic m1-5 receptorsubtypes; adrenergic receptors including α₁, α₂, β₁, β₂,; dopaminergicreceptors including D₁, D_(2a), D_(2b), D₃ and D₄, and D₅ ; serotoninreceptors including 5-HT₁, 5-HT_(1-A-D), 5-HT₂, 5-HT₃, and 5-HT₄ ;benzodiazepine receptors; opiod receptors including o, κ, and μ; andothers. Also preferred are receptors for hormones and growth factorswhich may, for example, include those for insulin; growth hormone;erythropoetin; neurotrophic factors, including but not limited to nervegrowth factor, ciliary neurotrophic factor, brain derived neurotrophicfactor, NT-3 and NT-4. Receptors for cytokines such as interferons, andthe interleukins are also preferred as are receptors for nonpeptidehormones such as thyroid hormone, and glucocorticoids. The methods andcompositions of this invention described herein may be adapted bymethods known in the art and applied generally to identifying thespecific binding surfaces of other pharmacological targets as well.

Other target surfaces for which active ligands may be identified includeextracellular, intracellular, nuclear or mitochondrial located solubleor membrane associated proteins, carbohydrates, lipids nucleic acids orcomplexes thereof which play a role in a physiological orpathophysiological process involving a predictable indication for whichone would like to have a drug based therapy.

The pharmacological targets according to this invention, arephysiological molecules, or combinations of molecules associated throughcovalent or non-covalent forces, which alone or in combination withother molecules, evoke a physiological or therapeutic response whenactivated by a ligand which binds the "active surface" of thepharmacological target. By "active surface" is meant the region of thepharmacological target which can bind a ligand, whether or not there arenative endogenous ligands for these sites, and translate that bindinginto a physiological meaningful response characteristic of the target.Where the response requires oligomerization of at least two separatemolecular entities by a ligand, binding to the active surface on onlyone of the molecular entities is insufficient to evoke the physiologicalresponse.

The active surface is comprised of specific atoms or other chemicalmoieties which participate in the binding of the ligand to thepharmacological target, for example by contributing to changes inenthalpy or entropy. The active surface of the pharmacological targetmay be small, capable of being bound by a single monovalent ligandhaving a molecular weight of less than about 1000 daltons; or large,requiring a multivalent ligand for binding to a plurality of bindingsites which contribute to the active surface. Multiple binding sites maybe present in a larger binding domain in a single region of thepharmacological target. Alternatively, multiple binding sites may bepresent as separate non-contiguous regions which may be bound by aligand capable of spanning the pharmacological target to simultaneouslybind the different binding sites of the target. In addition, bindingsites may be present on two or more molecular entities, which may be thesame or different, and which require oligomerization by binding to amultivalent ligand.

Growth Factors (GF), including NGF, EGF, FGF, interleukin (e.g. IL2, 4,6) interferons, insulins and many other extracellular biosignals alongwith their respective receptor targets apparently contain multipletarget binding sites. Such protein signals are in the order of 20-1000 KDaltons and exist as monomers or homo- or heterodimers or more complexmultimers, which encompass surface areas of tens of thousands of Å².Estimates of the surface area of such endogenous ligands and receptorswhich are occluded by their association ranges from 500-1600 Å². By theabove definition, each ligand has ≧2 binding sites and each receptor has≧2 corresponding binding site which are discontinuous andnon-overlapping with each other.

II. Use of Recombinant Antibodies rVab's as Scanners to Identify ActiveSurfaces

This invention identifies and characterizes active surfaces byconstructing and using a sufficiently large repertoire of diverseligands capable of "scanning" the surface of pharmacological targets andbinding to their active surfaces. Confirmation of binding to activesurfaces is accomplished according to this invention by monitoring achange in function of the pharmacological target or by monitoring abiochemical or biophysical change which reports binding and/oractivation of the pharmacological target or receptor on the target.

Antibodies have most of the above required attributes and can berecombinantly engineered so as to acquire unique attributes required foruse in this invention. It is well known that antibodies occur which areneutralizing and therefore by definition antagonistic in that theyprevent, competitively or allosterically, the binding of signal toreceptor, or receptor activity.

Antibody epitopes in protein targets range from a few amino acids toabout 20 amino acids and cover from hundreds to thousands Å² of targetsurface. In addition, epitopes can comprise sequential or noncontiguousgroups of amino acids. However, it is equally clear that antibodies canrecognize organic epitopes which are relegated to much smaller volumes,(i.e., <50-200 Å²) as are those associated most frequently with smallorganic haptins (i.e., clinitrophenol or morphine). As antibody affinityand selectivity can be equal with both large and small epitopes, it isassumed that anti-target rVab antibodies will have landscape recognitionsurfaces which range over all of these dimensions.

A. Use of rVab Libraries

The repertoire of different ligands for scanning the pharmacologicaltarget according to this invention is provided by an antibody librarycomprising recombinant Fab fragments, or portions thereof, constructedto present a sufficiently large repertoire of different identifiablestructures, some of which will be expected to bind and, depending onwhether concurrent binding to multiple sites is required, activate thepharmacological target. These active antibodies are identified asspecific members of a library which may be considered to scan the entiresurface of the pharmacological target and possess the desired compositeactivity profile for the binding site. According to this invention, therecombinant antibodies used with this invention are referred to as"rVab" to indicate that they are constructed using recombinanttechniques and are made as libraries which incorporate diversified aminoacid sequences in one or more regions of the antibody associated withtarget recognition or binding.

Where the pharmacological target comprises multiple binding sites on onemolecular entity, or requires oligomerization of at least two moleculesto form a single binding site with contributions from the individualsubunits, or requires oligomerization of two or more molecular entitieswhich each bind to the ligand at a different site, activity will only beobserved using antibodies modified according to this invention tocontain at least one additional separate binding entity. In thepreferred embodiment of this invention, the separate binding entitycomprises at least one random sequence of amino acids having a structureappropriate to bind a binding site not bound by the antibody's variableregion. In some cases two such random sequences of amino acids would berequired although it is contemplated that additional sequences may alsobe required. Additional binding sites on rVab can also be provided bymore or less complicated protein based structures including smallerpeptides, larger proteins including intact enzymes or even anotherantibody, in structures described in the literature such as diabodies(Winter et al. 1994). Additional peptide sequences which may be used toadd additional binding sites preferably are between about 5 and 30 aminoacids in length. More preferably such sequences are between about 6 and12 amino acids in length. Most preferably, such sequences contain 8amino acids.

An antibody identified as recognizing the binding site is simultaneouslyor sequentially further characterized by determining its selectively andactivity for the pharmacological target. To streamline an rVab selectionprocess for more than one target attribute, target specificity (T) andsome of the activity (A) testing may be simultaneously characterized.

The order of isolating Vab for A⁺ and S⁺ can be varied, most oftendepending upon which is the more difficult attribute to find amongentities which modify the target of interest. For example, ifselectivity among highly homogeneous target members of a single familyis the critical missing attribute of existing agents, S⁺ could bedetermined first, or after isolation of the population which is A⁺.

Although antibodies which recognize (i.e. bind) the target's landscapein such a way as to modify its function make up a small percentage ofthose capable of passively recognizing the target (i.e. not modifyingits activity), their presence is likely because of the size anddiversity of the rVab library of the invention. In addition, activeantibodies would also be expected to be present which have theadditional desired attribute of specificity for that target. Furthermorean embodiment of this invention includes that the biological suitcases(i.e, phage or bacteria) used to individually package each rVab librarymember allows their recoverability after a biological replicative cycleeven if present in the original library in rare copies.

This invention utilizes recent advances in molecular biology which allowthe generation and manipulation of sufficiently large and diverse V (VHand/or VL) region libraries, along with both minimization and directedsecondary diversification of their CDRs and CSRs to allow selectedrVabs, when labelled, to act as reporters and affinity selectors inassays which identify potential active ligands. Such active ligands arepreferably small organic molecules which are useful as drug leads or astherapeutics themselves.

B. Use Of rVAB Members To Identify Small Organic Molecule Replacements(SOMERS)

At least two methods are provided for identifying SOMERS based on theidentification of the recombinant antibodies (rVab's) possessing theattributes of T (specificity/potency) S (selectively) and A (activity).According to one method of the invention, these [rVab T+ S+ A+] scannersare converted to reagents for reporting the presence of other ligandscapable of binding to the active site on the pharmacological target.Conversion to reporters is accomplished by labelling the active scannerswith a detectable label. The reporter rVab fragments may then be used inclassical competitive binding assays to identify SOMERS. For simpleactive surfaces, single SOMERS represent active small organic molecules,while for complex active surfaces containing more than one ligandbinding site, corresponding numbers of SOMERS, found in the fashiondisclosed by this invention, are covalently coupled together torepresent the active small organic molecules.

In another embodiment of this invention, SOMERS are identified based onthe collective attributes of an ensemble of active rVab scanners whichhave been characterized as T+, S+ and A+. By providing a sufficientlylarge repertoire of antibodies, multiple antibodies possessing thesedesired binding attributes are expected to be identified. Commonstructural features of this ensemble of scanners possessing the desiredCAP attributes are then used to construct a model ligand for binding tothe active surface of pharmacological target. By combining structuralfeatures of multiple antibodies identified as being active for aspecific pharmacological target, biologically enhanced ensemblepharmacophores ("BEEPS"), i.e., drug models, may be derived which maythen be used to identify small organic molecules as drug leads ortherapeutics. This molecular model, BEEP, then serves to provide a basisfor screening chemical databases to identify SOMERS either by electronicscreening of available chemical data bases, or as a basis for rationaldrug design to synthesize SOMERS expected to possess the combinedattributes of specificity/potency, selectively and activity. This solvesthe prior art problem of access to all compounds within a chemical database, decreases the time needed for screening and amount of manpowernecessary, and could eliminate screening if used to direct a syntheticchemistry effort to create SOMERS.

III. Recombinant Antibody Libraries Provide Sufficiently LargeRepertoires of Different Ligands to Identify Active Surfaces

A. Function Of Recombinant Library

The objects of the invention are provided by a process which makes andthen uses separately and/or in batch mode, combinatorial repertoirelibraries of variable regions (VH and/or VL) of recombinant antibodies(rVab) to scan the surface of a pharmacological target so as to identifyand select those which have a desired potency, specificity, selectivityand activity profile. These four attributes are collectively definedherein as the compound activity profile (CAP). Members of the librarypossessing the desired attributes are then grouped according to thelocal surface domain recognized. By using a sufficiently large anddiverse library as described herein it is expected that essentially mostif not all relevant active surface of pharmacological targets should beidentifiable using the method, of this invention. In addition, becausethe library is recombinantly made in a random fashion and selected invitro, recognition of sites which would not otherwise be detected asnon-self, or antigenic, or immunogenic, should occur using the rVablibrary described in this invention.

In addition, the objects of the invention relating to discovery of threedimensional shapes of surface areas are provided by use of the activerVab's of this invention as reporters of target structure. As describedbelow, these rVab reporters are constructed using VH and VL domainswherein the CDR regions which may be diversified are contained in aframework of an Ig (or Fab) having a three dimensional structure whichhas been determined by crystallography has CDRs which contain the knowncanonical structures (CSRs). Such structural information about rVabs fora given delimited active target surface domain allows for the molecularresolution and deduction of the essential elements of the rigid organicstructure of the constellation of critical amino acids constituting theactive target surface recognition portions of the ensemble of activerVabs and thereby provide the essential elements of the rigid organicstructure of active SOMERS which can bind with specificity to and modifythat target.

Construction of the BEEP requires PCR determination of the amino acidsequences of rVab CDR, CSR and some framework residues in these activesurface scanners through a process which uses computers and geneticalgorithms. It is also possible that with sufficiently large enoughactive rVabs, the information obtained in the above manner will enableresolution of the active surface of the targets. This process providesthe objects of the invention related to electronic screening for SOMERSby combining common structural elements in computational packages calledbiologically enhanced ensemble pharmacophores (i.e., BEEPS).

The recombinant antibodies used in accordance with this invention alsoprovide an improvement over the prior art of typical labelledtarget-reporter binding assay screens. One improvement comprisesobtaining via recombinant molecular biology technology, antibodyvariable regions (V) in sufficient numbers, with sufficient affinity anddesired activity so as to identify those members of the library whichfunction as surface reporters capable of recognizing active targetsurfaces, modulating the target through these recognition sites anddistinguishing its target from among closely related targets(selectivity).

B. Size Of Recombinant Library

In order to have a sufficient likelihood of identifying the activesurface of a pharmacological target, the recombinant library preferablycontains at least between about 10⁹ and 10¹⁴ entities. Preferably thelibrary contains between about 10¹⁰ and 10¹³ entities. Most preferably,the library contains about 10¹² entities. The specific size of thelibrary required to provide a reasonable likelihood of identifying theactive site will depend on the overall surface area of the targetsurface and the surface area of the binding domain to be identified. Thesurface of most targets is of the order of 50,000-100,000 Å₂, with eachligand binding domain encompassing from about 100-200 Å² to about1,000-2,000 Å². As each rVab covers only about 20-40 Å² of surface area,one requires about 2,000 rVab's to cover the target landscape, and atleast 10 times that (2×10⁴) allowing for overlapping recognitiondomains. Another increase of two orders of magnitude (2×10⁶) allows forappropriate surface interactions which produce specific agonist orantagonist action. Another 100 fold increase allows for such rVabs to berecoverable from the library upon batch analysis. An additional 10⁴--10⁴ fold increase allows nanomolar affinities and agonistic activity.Accordingly, the preferred useful surface scanning libraries have on theorder of about 10¹² entities.

It is recognized that antibodies have the ability to distinguish amongclosely related targets. Accordingly, recombinant libraries possessingsufficient numbers of entities are reached according to this inventionby constructing recombinant libraries comprising variable regions ofeither, or both light (L) or heavy (H) chains which are modified orunmodified and which may or may not be expressed in combination with aconstant region. These libraries may be selectively varied not onlyduring their original construction, but also after the initial round ofselection for any one or all of the three composite profile activitiesof target binding, selectivity and activity. Such secondary additionaldiversification as well as secondary simplification may be carried outby combinations of primer based PCR or oligonucleotide insertion atconvenient restriction sites. Furthermore, the secondary variations maybe localized to each of the 6 CDRs (i.e. the three in VL and the threein VH) or any particular combination or singular location. Variabilityis introduced in the CDR's by modifying the CDRs to contain random aminoacid substitutions of positions involved in contact with the target. Thepositions of variation, including further diversification orsimplification, are preferentially those within the CDR which do notalter the CSR structure of that region and are known to those skilled inthe art. The number of amino acid positions to be diversified isdependent on the number of active rVab members desired to be obtained.Thus, if an insufficient number of members are identified, the librarydiversification can be increased by diversifying additional amino acidpositions in a CDR as described below.

Given that there are twenty naturally occurring amino acids,diversification at a single amino acid position results in about 20different potential antigen binding (touch) sites. By diversifying attwo amino acid positions in each of the 3 VL CSR, 2 VH CSR, and the oneVH CDRH3 which are randomly combined into VH:VL pairs by the invention,one obtains a diversity in the rVab library of ≧10¹⁸ members (see FIG.4). Since a given phage library can package about 1×10¹⁴ members,several libraries are preferably constructed and packaged in phage tocontain the entire population of diversified members. Although, it ispreferable to diversify two amino acids in each CDR as shown in FIG. 6,other combinations are possible. Randomization in only some of the CSRsand one CDR allows for library sizes approximating 10¹² such that onephage rVab library could contain multiple copies of each diversifiedmember. In addition, three or more non-essential amino acids in a givenCDR may be diversified (see, FIG. 5 for non-essential amino acids)preferably with a corresponding decrease in diversification of aminoacids in other CDRs so as to maintain the total size of the librarywithin an attainable number. Resultant libraries of 4×10¹² members canbe approached using, for example, bacteriophage as vectors. A singlerVab library of this invention of at least about 10¹² members,independently of how diversity is obtained, provides enough surfaceprobes with the minimum CAP at the target to allow identification ofmost active surfaces of interest.

An advantage of this invention over prior art screening methods is thatit scans the entire available surface of the target for active surfacesand provides active surface reporters. This allows for identification ofactive sites and SOMERS for targets without endogenous signals(endogenous ligand) and at target surfaces not used by naturalendogenous ligands but which result in modulation of that target. Thelatter surfaces, referred to as allosteric surfaces, are of two types:those without activity in the absence of endogenous ligand binding totarget (i.e., cryptic allosteric sites); and those having activity ontheir own and yet are still able to modify the action of an endogenousligand (i.e., active allosteric sites). Obviously, the larger the targetsurface under scrutiny, the greater the opportunity of findingappropriate active surfaces. As endogenous ligand contact surfacesprobably represent some 10% of total target surface area, includingallosteric surfaces greatly increases the surface area underinvestigation.

The use of recombinant libraries also provides a means of reducing orincreasing the number of complementary determining regions (CDR) withinthe variable domain of the rVab necessary to confer desired CAPattributes to the rVab. Thus, one can attain a minimal active CDRcomplement. Alternatively, large scale randomization of up to most ofthe amino acids within the rVab CDRH3 domain may be used to increase thepopulation of active rVab from which to identify the best rVab reporter.For example, if the initial library screened does not possess memberswith the sufficient constellation of CAP attributes, secondarydiversification of the best candidates, by a number of proceduresincluding PCR and various in vivo and in vitro mutagenesis systems knownto those skilled in the art, and then recycling through the originalidentification and selection procedures described below, may be used torecover an antitarget rVab with a full complement of the desired CAPwhich might have been too rare to be found among the original antitargetrVab library. In addition, by identifying and sequencing active rVab CDRcomplements one may also obtain accurate and detailed structuralinformation useful for modeling the essential elements of active SOMERS,i.e., as in BEEPS.

C. Affinity of Recombinant Antibodies

The rVab of the invention are used to detect and characterize activesites by providing information related to their structure, and/or tofunction as reporters in competition assays to identify SOMERS.Accordingly, the affinity of the rVab's useful in this invention shouldallow for one or both of these functions. If the rVab is used only todetect and characterize the active binding site or to contribute indeveloping a BEEP, its affinity may be high and a slow dissociation rate(i.e., half time of dissociation, preferably between about 5 and 30)would be suitable. However, the affinity of the rVab's useful toidentify SOMERS for a pharmacological target should not be so high as toprevent dissociation and competition for use in competition assays.Preferably, this affinity will be in the range of from about 0.01 toabout 100 nM. More preferable the affinity will be between about 0.1 toabout 30 nM. Even more preferably the affinity will be about 0.5-10 nM.Most preferably, the affinity will be between about 1-5 nM.

D. Characterization of Ligand And Target Binding Sites

Binding domains on a signal are referred to as ligand attachment sites(LIGATTS) and those on the target as target attachment sites (TARGATTS).Where each is protein in nature, both can be defined as the surface areaof the entity made up of contiguous (e.g., amino acids n and n±1) ordiscontiguous (e.g., amino acids n and i where i is not n±1) elements soconfined in space as to be accessible and in contact at the same timewith the surface of the other partner in the complex so as to contributeto the binding energy of that interaction. Where there are multiplebinding sites, by our definition, each TARGATT domain forms contactpoints with amino acids on the signal and one SOMER would not beexpected to encompass two LIGATTS. Where endogenous ligands arenonproteinaceous, other compound building blocks would replace the aminoacid as the unit entity.

The sizes of LIGATTS and TARGATTS are quite variable. We havearbitrarily confined TARGATTS to the volume which can be encompassed bya synthetic small organic molecule replacement (i.e., SOMER) of lessthan about 1 kD. This TARGATT size, is practical and modeled by theopiate receptors' attachment site for its 30 amino acid endogenousligand, endorphin, which easily binds morphine (<600 D) and all of thepharmaceutically known opiate analgesics, with nM affinity and is fullyactivated by their attachment. Identification and characterization oflarger TARGATTS is considered within the scope of this invention as suchsites should also be recognized by members of rVab libraries of thisinvention.

E. Association of Activity A⁺ With Binding of rVab

An important feature of this invention is that the rVab's which areidentified as possessing the desired CAP attributes and in particular,activity at a target, function to create a linkage between binding to atarget and activity at that target. Accordingly, once an rVab isidentified which is both T⁺ and A⁺, that rVab may then be used toidentify other ligands which are also T⁺ and A⁺ based on competitionbinding assays alone.

Several methods are available to initially provide a connection betweenbinding and activity of a rVab. In a preferred method, an active surfacefor a target is associated with a secondary biochemical response whichmay be detected upon binding of an active ligand at the active surface.Such biochemical responses may include changes in affinity of the ligandor allosteric ligands, oligomerization with other subunits,phosphorylation state, ion flux, etc. For example, and as discussed morefully below, the changes in agonist affinity of a receptor coupled to Gprotein based on the presence of a guanine nucleotide can provide thenecessary linkage between binding and activity.

Also, as discussed in U.S. Pat. No. 4,859,609, which is incorporatedherein by reference, receptors may be expressed as fusion proteinscomprising the ligand binding domain of the receptor fused to a"reporter" polypeptide which undergoes an assayable change inconformation or function when the active ligand binding domain of thereceptor binds to an agonist or antagonist.

IV. Method of Identifying SOMERS

The method of obtaining small organic molecules (SOMERS) which areactive at pharmacologic targets is summarized as comprising thefollowing (See FIG. 1):

Stage I (a): Construction of the scanning rVab library.

Starg I (b,c): Identification of rVab's which bind and activate target.If target is a multivalent site requiring attachment at two sites, pairsof rVab's are identified using rVab-peptide scanners to detect activity.

Stage II: Use labelled rVab's as reporters to detect SOMERS or MULTIMERS(i.e., DISOMERS).

Stage III: Create BEEPS from composite of structural information derivedfrom rVabTSA+ for screening or synthesizing SOMERS or MULTIMERS.

A. Construction of Scanning rVab Library (Stage 1a)

Molecular biology technology is used to construct a limited number oflarge combinatorial libraries of recombinant antibodies (rVab libraries)wherein the VL and VH CSRs and CDRH3 occur within each library within asingle Ig VH and VL framework, respectively, and optionally attached totheir respective constant region (CH1 and CL). An antibody whosestructure has been determined by crystallography is preferably used toprovide the framework for construction for these rVab libraries.Antibodies of undetermined structure can also be used for libraryconstruction and identification of active rVabs (i.e., Stage 1 abc,FIG. 1) useful as reporter rVabs to detect SOMERS and other MULTIMERS(Stage II FIG. 1) according to the process of the invention, but onlyantibodies of determined structure can be used in creation of BEEPS,(Stage III, FIG. 1).

In the preferred embodiment of the invention, antibodies of solvedstructure are used to create the original rVab library. In anotherembodiment, one or two of the isolated active rVabs for a given targetare subsequently crystallized and the structure determined to allowtheir use in Stage III. The later is useful as it allows use of thenewly published sequences of the human VH and VL genes [Tomlinson et al.1992; Williams and Winter 1993; Cox, Tomlinson and Winter 1994; Nissimet al. 1994; Tomlinson et al. 1994] for Stage III work.

In all cases, the rVab libraries constructed by the process of theinvention have a sufficient number of diverse members to encompass animmunological antigenic repertoire approaching man's natural one or aremade from human VH and VL genes [Roitt, 1991; Nossal 1993; Griffiths etal., 1994] which are capable of recognizing an enormous diversity ofsurfaces including but not restricted to proteins, nucleic acids,carbohydrates, lipids and organic haptens.

There are basically three sources of genes to be used as the startingmaterial for construction the rVab libraries.

a) the published data on cloned and sequenced antibodies;

b) the antibody clones themselves, carried in various cell types,including hybridomas, spleen cells, bacterial plant cells, yeast andviruses, on various DNAs including Plasmids, phagmids and chromosomes;and most recently

c) the published sequences of a human repertoire of VH and VL genes[Roitt, 1991; Tomlinson et al. 1992; Nossal 1993; Williams and Winter1993; Cox, Tomlinson and Winter 1994; Griffiths et al., 1994; Nissim etal. 1994; Tomlinson et al. 1994].

Most of the sequence information is available in at least two databases, i.e., the Brookhaven Protein data base and that of Kabat at NIH(which is also available in text form) [Kaba et al. 1991]. The structureof the majority of the crystallized antibodies is also available fromthe Brookhaven Protein data base. Listings of such crystallizedantibodies are presented in Example 1. An example of an antibody whichhas been crystallized to determine its structure is described in (Tulipet al. J. Mol. Bio., (1992) 227:149-150).

In the preferred embodiment, the antibody sequence is obtained first andis the starting point of rVab library construction using the followingsteps to construct the rVab library. The order of steps may be varied tosuit particular circumstances.

I. Selection of Parental Fabs of Known Crystalline Structure as rVabLibrary Framework Templates

II. Creating the Nucleic Acids Encoding the Heavy and Light Chains(rVHCH1 and rVLCL) for ABXXX rVab.lib.

Step 1a): Construction of 5'VL Section

Step 2: Diversification By PCR

Step 1(b): Construction of the MIDVL section

Step 1(c): Construction of the 3'VL section of rVL

Step 3: Ligation

III. Construction of the Constant Regions of ABxxx

IV. Construction of rVHCH1.lib (FIG. 8) Construction of 5' Half of theVH Region Construction of the 3' Half of the VH Region

V. VH and VL Library Sizes:

VI. Construction of the rVab.lib (the VHCH1lib×VLCLlib Combinatoriallib.) (FIGS. 11,12,14)

Step 4: In vivo recombination of VHCH1 and VLCL genes

Details of the Individual Steps for Expressing the rVLCL.1.6 andrVHCH1.L.b by CRE-LOX RECOMBINATORIAL FORMATION

VI. Step 5--Generating Phage and Displaying the rVab.lib on PhageSurfaces (FIG. 14)

The critical steps are shown in FIGS. 7, 8, 11 and 14 which describerespectively the construction of rVLCL and rVHCH1 libraries, theirpairing in the rVab library, and finally their expression attached tothe surface of phage as functional complexes.

Both construction of the rVLCL and rVHCH1 libraries follow a similaroutline wherein:

a. a limited number of oligonucleotides are synthesized containingconvenient restriction sites and which cover both ends, and in one casethe middle domain, of the V region,

b. the oligonucleotides are ligated together,

c. PCR is used to append missing and junctional regions as well asprovide the means of randomization of amino acids at defined positions,

d. the completed rVH and rVL libraries are ligated to appropriateconstant domains wherein one library is placed within a plasmid and theother phagmid, and

e. the rVH and rVL libraries are combined in vivo by the CRE-LOXrecombinase provided by coinfection by P1.

Following this outline, rVab libraries of about 10¹² members areconstructed.

In other embodiments,

a. the VH and VL genes, without constant regions, encoding an antibodyof known structure are cloned via PCR to obtain the sequences encodingthe VHCH1 and VLCL sections of the lgs using methods known to those inskilled in the art, and

b. the Vs may then be altered via PCR to remove unwanted restrictionsites, and develop convenient restriction sites bording the CSR and CDRdomains.

c. selectively randomized oligonucleotides with appropriate endpositional restriction sites may be used to replace each of the 6 CDRregions having appropriate matching restriction sites in the basic Vframework to allow directional cloning. These oligonucleotides vary inlength (i.e., n, n+1 and n+2) to match the known CSR and some lengthchanges in CDRH3 and contain all of the amino acids at one or twopositions within each CDR most often involved in antigen contact.

In the preferred and other embodiment, with 2 amino acid randomizationswithin each CSR and CDRH3 and three different lengths of CDRH3 used, thenumbers of diverse members in the final rVab LIB (i.e., rVHCH1×rVLCL)reach 10¹⁸ (see FIG. 4 for details).

1. Sources of Frameworks

Frameworks in which the optimally diversified CSRs and CDRH3 are clonedinto may be derived from antibodies of known structure.

Frameworks may be chosen from antibodies which present the canonicalregions in different orientations with respect to the C region. Thus, itmay be desirable to prepare multiple rVab libraries on differentframeworks to maximize different special orientations of the CDR's.

Frameworks may be chosen which will favor binding over small to largesurface areas. As discussed above, a small surface area would cover anarea of about 200 Å², a medium surface area about 750 Å² and a largesurface area about 1500 Å². Examples of antibodies which can provideframeworks for these three different size targets are found among theplanar, cavity and grooved type antigen recognition domain present invarious antibodies of known structure (FIG. 3 respectively). Frameworksmay be chosen simply based on the shapes of the antigen recognitiondomain or in combination with other structural factors.

2. The Expressible Vab Region Construct

Preferentially, construction may be done in one of two general typevectors,

a. fd and M13 (Pharmacia, USA [Smith, 1985; Scott and Smith, 1988;Parmley and Smith 1988; Cwirla, et al., 1990, McCafferty, et al. 1990;Winter and Milstein 1991; Waterhouse, et al. 1993, Recombinant PhageAntibody System Instruction Manual, Pharmacia P-L Biochemicals, USA].

i. the inserted V(H and L) with CH1 at the carboxy terminus preceded bythe lac promoter and a ribosomal binding site [RBS], an export leadersequence in front of gpIII phage coat protein or PelB, a cloning sitefollowed by either an in frame linker and then gpIII, or a double set ofsuppressible termination codons.

ii. the VH or VL without CH1 or CL or with partial NH2 terminal constantregion amino acids may be preceded by the lac promoter -RBS-PelB- withinternal cloning sites allowing in frame ligation of VH at both 5' and3' ends and followed by -C(H or L) and either an in frame linkage togpIII or two suppressible termination codons.

b. immunozapII (lambda) Stratacycte, CA, [Skerra, and Pluckthun 1988;Mulinax, et al. 1990, ImmunoZap Cloning Kit, Instruction ManualStratacyte Corp. CA USA; Kang, et al. 1991; and Barbas, et al., 1991].

i. as above for V region, with and without intact CH1.

ii. as above for V region, with and without intact CH1. Expression ofSingle V(H, L) -C(H or L).

Expression of single V(H or L) -C peptides may be used to confirm properconstruction of the V regions, or rVHCH and rVLCL libraries, beforeeither expression as mature VC (rVHCH or rVLCL) or CRE-LOX recombinationand phage expression. fd (M13) or Lambda expression is induced withglucose as described in Pharmacia (USA) Kits or the Stratacyte (CA)system Lerner. The product may be identified with CH1 antibody (standardElisa technology known to those skilled in the art) either with fd asphage displayed molecules, or with lambda after expression induction,and generation of periplasmic located molecules. When using phage, theinduction of the lytic cycle may also be used to determine the ratio oflambda to intact rV as an indication of size of library. With fd, onecan assay antibiotic (e.g. ampicillin) resistance colony forming units(cpu) transfer from within fd genome vs. the number of phage with rVdisplay attached to the viral surface. Dishes coated with viral or rVantigen may be used to provide information on the size of the rVlibrary.

In another embodiment, only the rVH and rVL domains are expressed andconnected through a flexible linker to form a single chain V regionantibody (termed scFv by Winter [Huston, et al. 1988; Bird, et al. 1988;McCafferty, et al. 1990; Hoggenboom, et al. 1991; Barbas, et al. 1991;Garrard, et al. 1991; Breitling, et al. 1991] which may be expressedusing phage display. The expressed V antibodies are fused to gIII on M13using a Recombinant Phage Antibody System Kits (Pharacia, USA),according to instructions provided the manufacturerer for construction,expression and detection.

c. General information on primer use and PCR.

To allow the library construction of various domains of rVH and rVL, andCH and CL as well, each primer includes a sequence encoding arestriction endonuclease recognition site. The sequence of the primerwhich contains the restriction site may be located within, partiallywithin, and sometimes precedes the section of the primer annealing tothe target Vab sequence. When it is present as an extension to thesequence homologous to the rV section under construction, it will notparticipate in annealing during first strand forward and second strandreverse synthesis but will participate in annealing subsequent PCRamplification cycles. Although not essential, the restriction sites (ateither or both ends) are such as to generate 3' or 5' overhangs to aidin subsequent ligation utilizing restriction enzymes which maintain theappropriate reading frames. Products of PCR may be isolated from thereaction mixtures by a variety of techniques known to those skilled inthe art. A number of restriction sites which have been successfullyencoded within rVH and rVL gene constructs for insertion in theavailable expression vectors are known to those skilled in the art andare available from manufacturers of IG expression systems and Ig primerssuch as Pharmacia (USA), Stratacyte (CA), and 5'-3' Prime (USA).

Insertion in frame can be into vectors containing sequences encodingother proteins to produce fusion proteins not only containing one ormore C constant regions, but also the coat protein gpIII and VIII of fdfilamentous phage, or transmembrane proteins to provide rVHCH or rCLVLanchoring for appropriate extracellular or phage displays.

3. Preparation Of rVabs With Multiple Attachment Sites

The grouping of active rVabs based on recognition of different targetsurface domains is simplified by using small peptides which cover in anoverlapping fashion, the liner amino acid sequence of the target. Suchgrouping simplifies the pairing of active rVab for a MULTIMER (e.g.DISOMER or TRISOMER) obtained from multivalent rVab-PEP libraries(example 3 and 4) as well as forms the basis of selection of active rVabfor conversion to reporters for simple SOMER identification.

Given that many antigenic sites are less than 12 amino acids, peptidesof 10-20 amino acids, made in overlapping fashion (i.e. amino acids1-15, 5-20 10-25 etc.) would provide most of the sequential targetepitopes. This would mean that for an average protein of 50,000 Kd,i.e., some 90 would be needed to cover the entire surface. For manypharmaceutical targets, mutagenesis and alanine scanning has providedinformation, known to those skilled in the art, of particular aminoacids, and small groups of amino acids which are involved in signalbinding and receptor activation. Such information is used here to reduceto a much smaller number the peptides needed to provide most of thedesired surface epitope information. Another possibility for targetfragmentation is the use of synthetic polypeptides, bought commerciallyor produced by biotechnology means, using commercially availableexpression vectors harboring specific sites for cloning and expressionof peptides in fusion with easily and quantitatively recoverableproteins.

4. CSR and CDR Diversification and Reduction

CSR and CDR Randomization: A preferred embodiment will be to usesynthetic oligonucleotides which vary at increasing number of amino acidpositions within each CSR and CDRH3 but which do not alter the CSR.Minimal randomization of amino acids would be to have only 1 positionwithin each CDR filled with all 20 amino acids. One could include up toabout 24 amino acid positions within the CDR H3. As the number ofpositions randomized increases, the total possible different rVH and rVLrapidly exceeds the practical limitation of 10¹²⁻¹⁴ on phage librarysize, and one has to limit the number to fit within the library sizethat is attainable. Increased randomization at larger number ofpositions can be accomplished by putting amino acids into classes, i.e.,basic, acid, hydrophobic, hydrophilic, etc., and then using only one ortwo amino acids of each group at each `randomized` position. Secondly,since not every amino acid within a CDR is involved in contact, one canidentify those which are most often involved in contact and focus aminoacid randomization at those positions. Lastly, one does not need to usethe same type or degree of randomization for all CSR and CDRH3 s. In oneembodiment, one could use only CSRH1, H2 and H3 for randomization as VHsalone have been published to have nM antigen affinity [Ward, E. S. etal. 1989].

In the preferred embodiment, randomization may be accomplished duringconstruction of the rVab library. In addition, secondary randomizationafter isolation of the initial active rVabs may also be utilized ifdesirable. Secondary randomization can be used to obtain a single, orpairs of missing attributes of the desired TSA CAP, or to increase ordecrease one or more present CAP attributes.

CDR Reduction: To determine the smallest target binding domain it may bedesirable to reduce the size of the potential rVab target bindingdomain. For CSR and CDR reduction there is the possibility of using onlyone VH or one VL, making PCR copies, cloning with primers which includeonly the first, first two, or last one or two CSR and CDRs within rVHand rVL, and subsequently ligating the constructs into parentalframeworks wherein the missing CSR or CDR has been replaced with astring of glycines (Winter EP 0 368 684 A1). After alteration eachlibrary may be retested for its new CAP. In another approach, one canstart with a preferred rVH:rVL pair and delete (again replacing eachwith a glycine heximer) a) one CSR or CDRH3 at a time (there being 5such possibilities); b) two at a time (there being 14 suchpossibilities); c) three at time (there being 9 of these); and d) fourat a time (there being 6 of these). With reduction in CSR and or CDRs,the potency of the altered rVab can be tolerated up to 30 nM, (thatrequired for use of the rVab in subsequent binding screens for organicreplacements). However, an affinity of 100 nM is tolerable in theminimal CSR/CDR combination if it is put through mutagenesis for potencyimprovements later on as such processes have been shown to produceincreases in binding affinity of up to two orders of magnitude [Bass,Greene and Wells, 1990; Marks, et al. 1991]. The reduction in numberplaces all of the critical contact atoms within the smallest number ofsemifixed domains making 3D modeling of critical atomic spacialrelationships easier by means known to those skilled in the art.

5. Expression of rVabs

a. Expression of rVab as a Phage Library.

In one embodiment of this invention, the rVab library is displayed onphage. This process best described recently by Griffiths et al. 1994).Methods for using phage display of antibodies have previously beenpublished (see, Ladner et al., International patent applicationWO90/02809; Winter et al. WO92/20791 and Huse et al. WO92/06204 whichare incorporated herein by reference) and some reagents are commerciallyavailable in kits.

In another embodiment, only the rVLCL is placed in a library forexpression as a bacterial plasmid construct (VLCL.bact) with a leaderwhich allows product release to the periplasmic space. This library isthen expressed and product is combined with either one of a rVHCHL orrVLCL phage displayed library to derive the two phage and one solubleprotein libraries. An anti-CL antibody attached to solid matrix may beused to harvest their VLCL protein library.

To identify members of the rVHCH1 phage library with one or more CAPattributes, the soluble rVLCL protein library is added to the abovephage library and panned for target surface recognition with targetprotein attached to a matrix (plastic, chromatographic or magnetizedbeads) in the absence or presence of competing proteins (see example 2)to derive rVHCH1:rVLCL protein T+ (S+) members. The phage containing therVHCH1 gene is harvested after allowing the phage to multiply withhelper (using commercial kits). Isolation and enrichment steps may berepeated as required. This library may be referred to asT+S+rVHCHhalfLIB. Assays for A+ may be then be done to obtainTSA+rVHCHhalfLIB.

The T+ (S+A+optional) rVHCHhalfLIB may then be cloned, in, for example,lambda and expressed as periplasm soluble entities. The library may thenbe mixed with the phage display rVLCL.LIB, and the above isolation stepsrepeated to obtain a T+ (S+A+)rVLCLhalfLIB. The specific methodology forthis procedure has been published by Lerner and group [Cabilly, et al.1984; Burton, et al. 1988; Huse, et al. 1989; Mullinax et al. 1990;Zebedee, et al. 1992; ImmunoZap Cloning Kit Stratacyte Corp. CA., andSurfZap Cloning Kit (instruction manual) Stratagene Corp, CA] and isherein included in entirety by reference. See below section onFunctional VH and VL combinations for details. The active rVHCHhalflibrary is cloned into pVHACCEPTOR (see FIG. 11) and the activerVLCHhalf library into pVLACCEPTOR. The CRE-LOX recombination system maythen be used to derive a rVab LIB combinatorial library which may betested for the TSA+CAP.

b. Isolation Of rVab Library Of Target Binders And Phage Display

This step isolates all rVab existing within the original library whichrecognize some part of the target's surface and form a complex withsufficient stability for isolation (i.e, target affinity <30 nM). Thosewith this recognition ability are termed T⁺. In the preferred method,the rVab genes are mixed and packaged in and displayed functionally onphage surfaces. Accordingly, rVab are displayed on the surface of phageand the phage are incubated with target surfaces. In other embodiments,the library number can be reduced by prior selection of activerVHCHhalfLIB and rVLCLhalfLIB which allows packaging and expression inbacteria, either as soluble or membrane anchored rVabs by methods knownto those skilled in the art using commercially available kits (e.g.Stratacyte USA) following manufactures directions.

As discussed above, the target can be any surface one desires to scanfor recognition by members of the rVab phage library. Permissibleincubation conditions, of which there are many known to those skilled inthe art, would include those which do not disrupt the vehicle packagingthe rVab, or inactivate rVab recognition of the target, nor preventdisplay of its target epitopes. In addition, in all cases therVab:target complex preferably is one which is quantitatively separablefrom free Tr-rVab phage packages.

After incubation of target and the rVab phage, there are many publishedmethods for separation of complexes known to those skilled in the artwhich are all based on the principle of having the target tagged(denoted Tr^(tagg)) in such a form as to allow its convenientquantitative separation from all reaction solutes. Preferably such tagsare inseparable or act as labels to follow the target:rVab complexesthrough separation procedures. Among such preferred tags are matrixessuch as agarose, magnetic beads and the surface of culture dishes. Inthese cases attachment of the target to the tag would have been madeprior to incubation with the rVab library. There are also non-matrixtarget-tags which allow target:rVab complex separation from solute andunbound rVab. Among such tags are fluorescent compounds, (for use influorescent activated sorting), biotin, (for avidin directed sorting)and polyhistine containing 6 residues [his 6] (for metal chelate columnchromatography) and very small antibody epitopes which are known tothose skilled in the art.

Incubation conditions can be varied extensively. Variations intemperature, time, pH, buffer and media additives are all to beconsidered as those attributes which influence target:rVab complexformation and stability in manners known to those skilled in the art.The preferred conditions here are phosphate, MOPS, Hepes or Tris bufferat about neutral pH (6.8-7.2) with 1% BSA at room temp. for about 4-6,up to about 6-12 hrs.

After formation of target:rVab complexes, any matrix bound rVab isseparated from unbound free rVab. In the preferred embodiment, thetarget is attached to plastic culture dish surfaces, and one of anynumber of rapid procedures, such as panning, is used for separating freeand target complexed rVab. The general approach of panning at differenttemperatures, pH and the presence of the antigen have been shown toallow isolation of rVab with controlled affinity.

After detachment from matrix or affinity associated tag, by proceduressuch as low pH or others known to those skilled in the art, therecovered rVabT⁺ can be recycled through the selection procedure or anyvariant thereof any number of times. Published panning and affinitychromatographic procedures have shown single step enrichments of 5×10²-10³ per cycle. Although, the number of cycles can be varied, dependingupon the enrichment found per cycle, the abundance of a particularrVabT⁺, the total size and diversity of rVabT⁺ recovered, 3 cycles ispreferred. Other number of cycles may be chosen based on recoveredrVabT⁺ characteristics such as S or A.

Isolation rVabT³⁰ members can be done with different types of packagedrVab expressing functional rVab, including phage packages or solubleentities as discussed earlier.

In this preferred isolation step, the rVab can have one of the followingfunctional forms Fv(rVCvh or vl only), Fab, or scFv as described below:

a. Single functional VH or VL without (Fv) or with associated constantregions (Fvc) for the V heavy (CHn) and Vlight (CL or k) genes or partsthereof. Both types of F can recognize targets using only three of thesix V region CDRs present in a natural Fab. In the preferred case, theseFvc genes are first packaged in fd phage and expressed with the C region(or some part thereof) attached, and in frame, being respectively a CHn,CL kappa or CL lambda) in which in all cases the constant regions, aredevoid of their C terminal cysteine. There are a number of CH regionsavailable including CH gamma, or delta, selected based on the requiredsolubility and known to those skilled in the art. These V(or VC) genescan be expressed as soluble entities with or without tags or, as in thepreferred case, fused, in frame, to one of the phage's coat proteins(i.e., gpIII) for functional display. These Libraries comprising only Vregions are termed rFv and may be expressed packaged in phage for phagedisplay. rFv phage libraries may be screened for members possessing CAPattributes of T, S and A and may be further diversified as describedabove. Such libraries with entities containing a reduced number of CDRor CSRs may be derived as part of the secondary simplification processwhen there are a very large number of active rVabs or whensimplification is desired to foster the development of a more accurateBEEP.

C. Functional VH and VL combinations (rVab):

These combinations have two V genes with, or without, partial or intactconstant genes. Although they may contain like members, the preferredcombination is one VH (or VHCH) and one VL, (or VLCL). In the preferredmethod, rVabT⁺ with the particular VH and VL couple are co-packaged in asingle phage, on a single piece of DNA, as two individual gene products.For each phage, either VH and VL, may be expressed as soluble proteinand the other attached to gpIII to cause surface phage display of theFab. This coupling and expression of VH and VL can be made with orwithout identifying separately the VH phage library and VL phage librarywhich can recognize the target when in the presence of a library ofsoluble VLCL protein or VHCH1 protein respectively (see, supra).

In one embodiment, the sequential procedure to obtain functional rVabsis as follows: Three individual libraries are made. Two of ≧10⁷ phagepackages each expressing and containing only one V gene (VHCH or VLCL)attached to phage for surface display. The other is of the same size butis made of VL genes expressed in lambda as soluble VL proteins which canbe harvested from periplasm of bacteria expressing the VL solublelibrary. First rVabs are then made by mixing the soluble protein librarywith the VH phage library in solution prior to testing for targetrecognition. This mixing allows all VL proteins present to complex withany one VH expressed on a single phage surface package to form a phageattached noncovalent (disulfide bonds excluded) functional rVab. Thisallows the formation of all possible rVab combinations. To this mixtureis then added the matrix associated target under study and afterincubation, and complex formation, all phage carrying a matrixassociated target displayed as part of rVab displayed on the surface ofphage are isolated, preferably by one of the above noted panningprocedures. Subsequent isolation of phage DNA gives an expressiblelibrary of functional rVHCH1 phage which can be T⁺ (i.e., the T⁺rVHCHhalfLIB).

All the VH phage library inserts, before or after phage amplification asneeded, are next excised via simple endonuclease restriction digestion,and directionally cloned into a lambda able to express inserts assoluble periplasm proteins. After induction as noted above, the proteinfrom the T⁺ rVHCHhalfLIB is harvested from the periplasmic space to givea protein library with the potential to form complexes with the entitieswithin the other original phage library, i.e., the rVLCL library. Aftermixing the soluble protein library and this phage library, as above, theT⁺ rVLCLhalfLIB is isolated, as noted above.

In the final step of this embodiment, a combinatorial library ofpackaged pairs of T⁺ rVab is produced in which individual packagescontain one VH and one VL pair of genes co-expressed as separateentities but associated together in functional rVab complexes. In thepreferred embodiment of this procedure, these two genes are combined viathe CRE-LOX recombinase system reported originally by Hoess [Hoess, etal. 1982, Hoess and Abremski, 1985; Hoess, et al. 1986] and recently byGriffiths et al. 1994, which are included herein by reference. Inanother embodiment, the package is also a phage, and expression issimilar to the preferred embodiment but in this procedure, thecombinations of rVHCH and rVLCL are made by excising and ligating invitro the DNA in a fashion which allows randomization of VH and VL pairsbut only one pair per DNA construct. These constructs can be phagmid orphage to allow either bacterial or phage expression of the rVab. Inbacteria the rVab are isolated and tested by protein lifts, whereas inphage, the rVab is attached to a surface protein for display and assay.Both methods have been published [Hoogenboom, et al. 1991; Kang, et al.1991; Waterhouse, et al. 1993; Figini, et al. 1994; Jespers, et al. 1994] and are commercially available in kit form (e.g. Stratacyte, CA). Thepreferred method is phage display of rVab.

The advantage of the embodiment in which active rVxhalfLIB areidentified before combining them into rVabs, is that where combinationsof VH and VL are made randomly from a preselected T+ active rVhalflibrary, the independent preselection of active VHCH T⁺ and VLT⁺ genesis likely to have reduced the number of active rVhalfLIB members to lessthan 10⁵⁻⁶. This reduction in number greatly increases the chances ofderiving within a single phage library of 10¹² members, which isattainable with the methodology disclosed herein, all possible activerVabs.

The procedure used to isolate single VH and single VL and pairs of VH/VLwhich recognize the target has the added benefit of being rapid, andcontrollable as to the strength and nature of Vab target binding that isdesired. By the procedures outlined, a paired rVabT⁺ (containing ≧ about10¹⁰ entities) can be generated.

The procedures discussed above result in the isolation of a) rVH or rVL,which alone do not need the other to recognize the target, and b) therecombinantly derived combinations of rVH and rVL termed rVabs and scFvwhich, in the later case, have rVH and rVL linked together by a shortpeptide chain and expressed as gpIII phage protein fusion products oreven as soluble entities. Additionally, rVab in which both V domains areof one type, i.e., either VH² or VL² are possible by this invention.VHVH Fab have been reported with increased solubility. Altering CH1 forCH delta regions or changing specific and identifiable C amino acids,could also facilitate expression of novel rVabs.

The basic and preferred technology for cloning individual heavy andlight chain variable regions either alone, or attached at their Nterminus to leader sequences, or parts thereof, or at their C terminusattached to a constant region, or parts thereof, and placement intosuitable expression vectors, transformation and expression in acompatible host cell in active form by recombinant DNA Technology aredescribed in the art. See, Huse WO92/06204; Ladner WO90/02809; WinterWO92/20791, which are incorporated herein by reference.

To achieve high yield and faithful cloning of each active IgG, secretionof protein either as soluble extracellular protein or in the periplasmicspace is suitable. In addition, protein may be expressed as anextracellular (or on the surface of phage) facing transmembrane ormembrane-anchored functional protein which allows spontaneousdimerization of heavy and light chain intact IgG or V domains.

Methods of cloning from naive or immunized animals, entire spleenrepertoires of Vab heavy (Vabh) and Vab light (V_(AB) 1) in theirnatural or random pairings to derive enormously diverse combinatorialrepertoire libraries are known in the art. [Huse, et al. 1989; Sastry,et al. 1989; Milstein 1990; Clackson, et al. 1991; Marks et al. 1991;Winter and Milstein 1991; Hawkins and Winter 1992; Hoogenboom, et al.1992; Lerner, et al. 1992; Marks et al. 1992; Winter, et al. 1994].

B. Identification of rVab's Which Bind And Activate Targets (Stage 1b)

In a preferred embodiment of the invention, pairs of VH and VL antibodydomains (rVab) are selected both as biological scanners of specifictarget surfaces and information reporters of activity related to themolecular 3D structure of the antibody site involved in surfaceinteractions as well as the molecular 3D structure of the activeelements of the binding site. This structural information is relevant toidentifying the minimum structure of the LIGATT, which would need to beincorporated into a SOMER or DISOMER, to reconstitute the CAP of theactive rVab and regulate the target in the desired fashion. Thisinvention identifies the unique ability of rVab when used as librariescontaining at least about 10¹⁰ members to identify those portions of atarget's surface connected to function in such a manner as toimmediately provide the tools necessary and sufficient for screening fororganic replacements at the target with a desired CAP. In addition, anembodiment of the process uses genetic algorithms to construct 3D highresolution molecular models of the shapes of organic molecules which canfit into the active target and regulate activity so as to electronicallyscreen for or synthesize via computer programs SOMERS or DISOMERS.

Active target landscapes are those surfaces connected to target functionas defined as those able, when occupied by a ligand, of influencingtarget activity. It is known that antibodies, in a wide variety offorms, e.g. Ig, Fab₂, Fab, or sFv (i.e., VH or VL alone), haveexceptional selectivity as well as high affinity for their targets. Thisinvention uses rVab which are identified as possessing the desired CAPattributes in two ways. Structural characteristics of multiple rVab'sidentified as possessing the desired CAP attributes are combined toproduce a composite structural map which is used to define a BEEP. Inaddition, individual rVab's which are identified as possessing thedesired CAP may be labelled so that they may be used as reports incompetitive binding assays to identify SOMERS, DISOMERS or other ligandsactive at the pharmacological target.

1. Identification of rVab's with TSA+ For Targets Having EndogenousLigands

The approaches to isolation and identification of Vab for targets havingendogenous ligands and rVab processing all TSA+ attributes, are dividedbased on two fundamental issues: first whether the rVab induced targetmodification is allosteric (alloA) or competitive (compA) with thenative signal (endogenous ligand) and second, whether the active surfaceis a simple or complex landscape found one or more different submits ofthe target. Target modification is considered anything which alterstarget activation by any means including native signal recognition(i.e., signal binding) and/or the signal transduction process directedby the active target. For example, the binding of ACh to the muscarinicsubtype 1 receptor and the interaction and activation of the Gi protein,respectively. In both cases, the process uses libraries already selectedfor, preferably by batch mode selection, target recognition i.e.,rVabT+, Batch mode selection is preferably than used to identify andseparate rVabT+A+ from those which are inactive under specifiedconditions. Libraries of 10⁶ to 10¹² individuals are used and theprocess is therefore applicable to rVab libraries which have both) THand VL chains, noncovalently (as Fab) or covalently attached (as scFv[Hoston, et al. 1988; Bird, et al. 1988; McCafferty, et al. 1990;Hoggenboom, et al. 1991; Barbas, et al. 1991; Garrard, et al. 1991;Breitling, et al. 1991] or diabodies [Holliger, Prespero, and Winter,1993] as well as those with only one V chain. By methods known to thoseskilled in the art, individual rVabTSA+ within an active rVab A+ library(LIB) can be simply and rapidly isolated, assayed, tagged and used toscreen various chemical libraries for SOMERS which compete with rVabA+for binding to the target.

For allosteric Vab-modulators, the presence of allosteric activitywithin a rVabT+ library is indicated by the occurrence of an alterationin the association between rVabT+ and the target induced by the bindingto the target of another entity. This entity could be the native signalor any known target effector entity. Examples of allosteric entitiesinclude such nucleotides as ATP for receptor containing kinases, or GTPfor G-protein associated targets, or a protein which couples to thetarget during signal transduction such as G-proteins, or even otherreceptor subunits.

a. Identification of rVabTSA+ from rVabT+ using allosteric modifiers

The isolation of rVabTA+ from rVabT+ is tied directly to the action ofthe signal at the target. In the preferred process, matrix-linked target(m-Tr) is mixed with the rVabT+ and incubated so as to allow m-Tr:rVabT+complexes to form. In general these are the same conditions used toisolate rVabT+ in Step I (b). After sufficient time to allow appreciablecomplex formation, which may or may not be sufficient to allow theinteraction to come to equilibrium, the temperature is lowered to about4° C. so as to trap bound rVab in the m-Tr:rVab complex by slowing itsdissociation rate. With the temperature at 4° C., free rVab is rapidlywashed away and the complex is resuspended in original buffer. Thisprocess is done quickly and uses a matrix such as, for example beads orplastic surfaces, and takes <1 min. For this process, preferentially onefirst determines or estimates the normal dissociation rate of rVabT+from the target. This may be determined by methods known to thoseskilled in the art. For example, in parallel reactions, the dissociationconstant (k-₁) for target (Tr) and signal are determined using either alabeled target (T*) and monitoring the dissociation of T*-rVabT⁺ -matrixcomplexes, or unlabeled target and following its release from the rVabT⁺-matrix complexes using anti-rVab constant region antibodies (oranti-phage antibodies) or by simply assaying phage in the supernatant ifa rVab phage library is used. The half time (t_(1/2)) for k-₁ at 4° C.for rVabT⁺ library from the target, for the entire population, is thendetermined.

With the t_(1/2) for k-₁ known, a new population of washed rVabT⁺-matrix complexes of the entire rVab library are formed at 4° C. andallosteric effectors are added in saturating concentrations. Half thepopulation is centrifuged to isolate the free rVabT+ members from thelibrary which remain in the supernatant within about the first minute(or ≦1/30th) of the population's dissociation t_(1/2). The remaininghalf is allowed to dissociate for about 10×t_(1/2), centrifuged and thepellet resuspended and allowed to dissociate for about another10×t_(1/2) to isolate the second population of free rVabT+. In bothcases, centrifugation is used to rapidly isolate the free rVabT+. In thefirst instance the free rVabT+ library is enriched for those rVabmembers induced to rapidly dissociate, referred to as rVabT+A+ allofast,while the second is enriched for those which have been induced todissociate slowly, referred to as rVabT+A+ alloslow. Each is thoroughlywashed. and then recycled through the above isolation procedure a secondtime. Such enrichment cycles are continued until a clear change inentire populations t_(1/2) for dissociation is seen at which time thepopulation is termed rVabT+A+ (fast or slow). Their numbers are thendetermined, if need be after amplification. If these populations are small, individual rVabT+A+ (fast or slow) can be isolated at this timeand assayed directly in subsequent procedures. If large populations areobtained, they can be analyzed in subsequent steps to isolatesubpopulations which have other desirable target attributes, e.g.specificity (S+) among one of a large number of target family members.

b. Identification of rVabT+A+ from rVab Library Using Competition Assays

The second approach to isolating rVab capable of target modification isused for the isolation of rVabT+, whether or not the S properties haveyet been determined, which are target regulators which bind to targetsat the same domain or at a domain overlapping with that used by thetarget's natural signal (nS) endogenous ligand. These are considered ascompetitors with nS for binding to the nS binding domain, and thereforeare competitive modulators, not allosteric modulators. Both agonists andantagonist replacements for endogenous ligand will be found within thispopulation.

This process requires the use of a high affinity nS which is labelled(nS*) and capable of rapid and quantitative isolation. There are manysuch labels possible, one is biotin, another, for example, is the smallantibody epitopes for which high affinity sera (or monoclonalantibodies) exists commercially. Methods of making such a labelled nSand the available epitope/antibody combination for protein signals andorganic molecules are known to those skilled in the art. Labelling is arelatively easy procedure for protein nS. For organic molecules it ismuch more difficult but in the preferred cases where labelling has notyet been done, non-neutralizing monoclonal antibodies or biotin will beused by methods known to those skilled in the art.

The preferred process of identification and isolation of competitiverVabT+ (S determined or undetermined) which is outlined here uses biotinas the nS label ("tag"). The process works similarly using otherlabelling tags such as iodination with ¹²⁵ I, or [³² p]ATPphosphorylation.

The biotinylated high affinity signal, nS^(tag), and the rVabT+ libraryto be tested (previously isolated and identified as T⁺) are combinedwith a soluble active form of the target (Tr) and incubated so as toallow formation of significant numbers of nS^(tag) :Tr as well asrVab:Tr complexes. The incubation conditions used here are thosepreviously used to allow binding of the rVab library to m-Tr as long asthese conditions also allow nS^(tag) binding to Tr. The temperature isthen lowered to 4° C. and all nS^(tag) and nS^(tag) :Tr complexes areremoved from solution with strepavidin (or another tag recognizercoupled to some matrix). The supernatant, containing T:rVabT+ complexesand free rVabT+ is affinity separated to isolate only Tr:rVabT+ byeither panning over anti-Tr antibody coated dishes or passed throughanti-Tr antibodies coupled to agarose. The anti-Tr antibodies used inthis step do not alter rVabT+ binding to Tr. Such antibodies are knownto often be those which have epitopes at either the amino or carboxytermini of the Tr under study or some other non-modulatory (i.e.,non-active) target domain. The population of rVabT+ bound to Tr insolution and obtained by association with anti-Tr antibody on their ownmatrix can be isolated and recycled through the above procedure anynumber of times for enrichment and amplification. This populationcontains all rVabT+ library members which bind to Tr at the binding siteused by the target's nS. This population is therefore made up of rVabwhich bind to the nS binding site and will be labeled rVabT^(+comp).Even though at this point these active rVabs are uncharacterized as toagonist or antagonist activity, their classification as active rVab isappropriate based on the definitions and disclosure of this invention.

Individual entities within these populations may be isolated, tested foragonist or antagonist activity using standard in vitro, cellular or invivo assays known to those skilled in the art, and/or labeled byprocedures known to those skilled in the art and used for screening foragonist and or antagonist SOMERS. Furthermore, where a labelled nS^(tag)exists for Tr, individual rVabT+A+compt will be tested for competitivemodification of nS^(tag) binding to T by methods known to those skilledin the art.

c. Isolation of rVabT+ Which Are A+ By Allosterically Modifying Targets

The next process outlines the isolation of rVabT+ which allostericallymodify Tr (i.e., are A⁺) by binding to sites which do not alter nSbinding but do alter the ability of the target to be active even fortargets devoid of native signals. In these cases, active rVab will beisolated by virtue of their ability to alter the association of T andsome component of the signal transduction system used by the target. ForG coupled receptors, that would be the GTP-G protein complex; fortargets with catalytic or stoichiometric enzymatic activity that wouldbe nonhydrolyzable substrate analogs; and for channels or transportersit would be ions, molecules transported, electrochemical gradients orother channel subunits. In these cases the isolation of this type ofrVabT+A+ would occur either by a) testing in batch mode limited sizedlibraries i.e., rVabT+A+ for agonist or antagonist action in vitro; orb) isolating in batch mode those which altered Tr activation, i.e.phosphorylation, binding of ATP or GTP, or binding of other proteinsinvolved in signal transduction as outlined above. Library members whichare T+A+ may be diluted and retested until single entities areidentified.

d. Identification of rVabT+A+ Pairs When Single rVabT+A+ Are NotIdentical

If no single allosteric or competitive rVab is found in cases where annS exists by one of the above approaches, the following procedures arecapable of identifying pairs of entities which, are both requiredsimultaneous as the necessary condition for modification of the target.In these procedures, the pairs of entities tested will be provided bytwo differentially identifiable rVab libraries or preferably one rVablibrary and another large and highly diverse library of identifiablemolecules. For targets with large protein signals, such as growthfactors cytokines, etc (i.e., >10,000 D) which may be expected to havemore than one LIGATT this dual modifier assay will be the preferredapproach in one of two general alternative forms.

The basic procedure will be described first using two differentiallylabelled rVab library as sources of the two paired modulatory entities.In addition to the rVab libraries there are both a labelled Tr (Tr*) anda labelled high affinity signal (haS) which are also recognizableindependently and separably from each other as well as from the rVab byhigh affinity probes. In each case, recognition of target occurs whetheror not these entities are part of any type of Tr complex but does notperturb the target's ability to bind haS* or rVab. For example, thelabelling epitope contained within the Tr* could be one which isrecognized by a high affinity Ig at sites commonly known to thoseskilled in the art as non-neutralizing epitopes. Large protein targetsare known to encompass such sites within internal peptide sequences, N-or C-terminus or unmodified or modified amino acids. These epitopes needonly be exposed during complex formation and non-active, i.e. unable tomodulate target binding of nS when occupied by recognition antibodywhich can be easily established in each case.

For signal labels, either biotin or an integral Ig epitope, are thepreferred label, allowing avidin- or Ig-agarose respectively, to be thequantitative recovery probe as long as the labels do not significantlyreduce affinity for the target. Other possible labels includeidentifiable peptides or protein sequences, such as substance P, partialHSV viral coat protein sequences, and enkephalin. The antibodies forsuch small epitopes or peptides could be either polyclonal or monoclonalIg, commercially available or rVab as procured by the recombinantmethods referred to for targets disclosed herein. Biotinylation ofvarious signals and testing for non-interference with native targetsignal binding to Tr is available by many methods known to those skilledin the art.

Using an Ig epitope labelled or tagged Tr (Tr*) and a biotin-labelledhigh affinity signal (haS), the identification and isolation of a pairof modulatory entities (in this example both are rVab) is initiated bycombining sufficient numbers of two previously isolated large rVabT⁺populations, each with a specific Ig epitope (epitope 1 for rVab1 andepitope 2 for rVab2), with the haS^(biotin) and the epitope taggedTarget (Tr*) to allow formation of the trimeric rVab1:T:rVab2 complexwhich does not bind haS^(biotin).

rVab1 and rVab2 may be added initially at a variety of about equalconcentrations from 10×1¹ down to 10⁻⁴ M. The lowest concentration atwhich target activation occurs will be used for subsequentmanipulations. The upper number is arbitrary but should theoreticallyexceed by about 30 fold the concentration needed for rVab1 or rVab2 tobind to Tr so as to saturate the site and prevent binding of haS*. Themixture is then allowed to incubate at room temp for at leastapproximately 6 hr, or overnight and then saturating amounts ofavidin-agarose is added and the mixture centrifuged and the supernatant,devoid of any free haS^(biot) or Tr:haSbiot complexes, is removed forsubsequent use. The supernatant, containing dimers of Tr:V_(AB) 1 andTr:V_(AB) 2 and the desired trimers of V_(ab) 1:Tr:V_(ab) 2 are thenpanned over anti-T Ig attached to a solid matrix or support such as forexample, plastic culture dishes or agarose column matrixes.

Identification and isolation of Tr complexes having both rVab1 and rVab2concurrently bound can be made by panning successively over matrixescoated with anti-rVab1 and then anti-rVab2 Igs. Phage displayed rVabsisolated by this procedure can be separated, amplified and then used forsecondary cycling through the above isolation procedure. Finally,individually purified phage are tested in identified combinations forcompetition of hastag binding.

In the above case, the two rVabT libraries (i.e., rVabT1, 2⁺) can beeasily distinguished for example by utilizing the CH1 domain of humanson one and the CH1 domain of mice on the other. Ig specific for humanand mouse CH1 are available commercially. Use of other constant regionsfrom one specie is also possible.

e. Use Of rVab-Peptide Libraries And Other Probes To Identify MultipleLIGATT Targets

i. Identification of First Ligand for a Multiple LIGATT Target

There are a number of variants to the above procedures in which thesecond entity of the pair needed to compete for haS^(tag) binding wouldnot be another rVab but instead would be a member of another librarycontaining diverse small organic molecules, peptides, nucleic acids,carbohydrates or even natural products. Excluding the possibility ofstearic hinderance, the frequency in the rVab library of entities whichbind to a target in a modifying manner (given their paired entity isalso present) should be no different than that for rVab which are ableon their own to bind to Tr surfaces and modify signal binding.Accordingly, rVab libraries of the size generated by this invention maybe used to identify both rVab members of the sought after pair. All ofthe libraries stated above having in excess of 10¹¹ members/ml should besuitable for use with this invention provided the frequency for eachbinding event is not less than 10⁻⁵. A useful library or pair oflibraries should contain sufficient members so that two binding eventswill occur simultaneously on the same Tr, the condition necessary forinhibition of haS* binding, at less than about 10⁻¹¹ and therefore bepresent at least once per reaction. If the frequency of each event isgreater, i.e., 10⁻⁴ or 10⁻³ then these modulatory complexes will occuras frequently as 10 to 100 times per assay. As the purification of anactive phage displayed rVab per cycle is 10⁻² to 10⁻³ then up to 4cycles may be needed to purify the active entity. To obtain one memberof the pair, one only has to purify from the final step, one of the tworVab entities. When other libraries are in use as the source of thesecond pair member, they need not be isolated at all.

ii. Identification of Second or Subsequent Ligands for Secondary LIGATTSof a Multiple LIGATT Target

Once one member (primary member) of the pair is identified, which in theabove case would be a rVab the isolation of the second is madestraightforward by using the first member, at saturating concentrationin all reactions. This simplifies to a search for a single entity, whichfor a rVab, would be done as outlined above. However, when one rVab of apair is in hand, one can search through a chemical as well as a rVablibrary for the second member of the pair of Tr binders which regulateTr activity when simultaneously bound to the target. Each member of thepair, particularly those which are identified as members of a chemicallibrary, are potential candidates as one half of a pair of small organicmolecules, one for each active surface domain required for targetregulation, which when covalently linked together would provide a singleactive organic molecule referred to as a DISOMER. Such DISOMERs would bevalid interesting drug discovery leads.

Another protocol for identifying an active pair, i.e., a pair which isnecessary and sufficient to bind to Tr in such a manner as to displacehaS^(tag), is to perform the original incubation of tagged target (Tr*),high affinity target signal (haS) and target binding rVab (rVabTr1 orTr2⁺) in the presence of excess labelled Tr* to reduce to a minimum thepresence of unbound rVabTr1 or Tr2⁺. If these incubations are done inthe presence of haS at about a 100 fold excess of the Tr-saturatingdose, the only rVab in solution will be those which has been competedfrom binding by haS. Accordingly, those rVab prevented from binding toTr by haS, should, with high probability, be those which can prevent haSbinding to Tr and are expected to possess the desired activity. As boundrVab can be separated from free rVab via panning over anti-Tr Ig (oravidin with a biotinylated Tr), upon such removal of all rVab:Tr*complexes, the only rVab remaining in solution will be those pairs whichwhen bound together, and possibly individually, prevent haS binding.Recycling of the supernatant additional times through such a paradigmwill eventually result in identifying the rVab pair or at least one ofits members if another type of ligand is used as the source of the otherhalf of the active pair.

iii. Use of rVab-Peptide as Surface Scanners

For signals such as protein hormones and growth factors, wheredimerization or timerization of identical (i.e., homoligomeric) ordifferent (i.e., heteroligomeric) receptor units is required forreceptor activation. This invention solves the problem in one embodimentby creating bivalent rVabs which allow for the isolation of bivalentactive rVab surface reporters capable of identifying each receptorsubunit endogenous ligand TARGATT attachment site. In this process,identification of bifunctional active surface reporters, proceeds bytaking a plurality of rVabs which have previously been identified asrecognizing either a particular limited surface of one of the target'ssubunits (i.e. are T⁺), or a larger number of one or two selected groupsof amino acids which are known to be involved with endogenous ligandbinding. The genes encoding these rVabT⁺ ligands are modified to encodefor a flexible amino acid which attaches in frame to one end of eitherthe heavy or light chain construct, a library of small random peptidesto create a bifunctional scanner (rVabPEP). In one embodiment, thepeptide is encoded by DNA used to that encoding the heavy or lightconstant domains. In another embodiment an rVab is expressed with atleast two peptides for identification of trimeric receptors.

In a preferred embodiment, a bifunctional scanner library consisting ofrVLCL and one rVHCH1 is constructed to identify rVab-PEPs whichrecognize an active surface consisting of two TARGATTS on the surface ofthe target. rVab-PEP are then isolated in batch mode and individualmember are subsequently identified as active competitors for endogenousligand binding. Such rVab-PEPs do not significantly bind the target inthe presence of excess endogenous ligand. These bivalent rVab-PEPs willthen prebound to target will prevent binding of the target endogenousligand which has been immobilized on a solid matrix.

For homodimeric receptors where each target subunit has a TARGATT whichbinds to the ligand (as per Growth Hormone Receptor, GHR), rVab-PEPwould be isolated. The rVab portion of a first active rVab-PEP is thenlabelled for use as a reporter to identify SOMER replacements for theLIGATT which resides within the rVab portion of the active rVab-PEPentity and recognizes one TARGATT on the surface of the receptor. Toidentify a second SOMER replacement for the second LIGATT of therVab-PEP entity, which resides in the PEP portion of the rVab-PEPentity, a second rVab without peptide is identified from the library ofactive rVab-PEP which competes for binding with the peptide portion ofthe first rVab-PEP. The process of finding the two rVab which correspondto the two LIGATT residing within an active rVab-PEP entity is referredto as rVab Pairing. The second rVab is then labelled for conversion to areporter for identification of SOMERS for the second LIGATT site.

Where the targets are heterodimers, the preferred approach is asfollows. The rVabT⁺ for receptor subunit surface I, are grouped basedupon recognition of common domains and/or surfaces containing amino acidknown to affect binding of endogenous ligand. These rVab's are thenexpressed as rVab-PEP as described above to generate a series ofbivalent ligands. Members of this rVab-PEP library which are displacedfrom target by endogenous ligand and which also displace endogenousligand from the target are selected as above for homodimer receptors. Alimited number (≦about 10) of rVab-PEPs with endogenous liganddisplacing activity at the target are then selected for identifying aligand for the second (II) binding site. An alternative selection methodfor identifying site I ligands is to select rVab-PEPs based on theirability to activate target. Activation may be detected as describedabove based on modification of an allosteric effector or on some otherdetectable change associated with receptor activation. For example,activation may be associated with self phosphorylation or dimerization.rVabs for the second TARGATT site on the second receptor subunit of theheterodimeric are identified in one embodiment, by expressing rVabs as arVab-PEP library using rVabs previously identified as being competitivefor the endogenous ligand at site II. The resulting rVab-PEP library forsite II is then tested for activity as described above and activemembers are isolated.

V. Identification of rVab which are Selective (S⁺)

In order to isolate those rVab which are selective for and distinguishamong closely related members of a target family or any target ofconcern (i.e. selective), the following batch mode selection proceduremay be used. The rVabT⁺ under investigation is mixed with matriximmobilized target (m-T) and allowed to form complexes in the presencesof soluble peptides, recombinantly obtained protein fragments or intacttargets whose identical (or related) sequences or conformations arefound in targets for which the investigator does not wish the rVab tobind. These sequences are typically between about 6 to 12 amino acids inlength and are present in the targets for other endogenous ligands ofthe same gene family. After sufficient time for complex formation therVabT⁺ still bound to matrix are isolated by panning and preferablyrecycled 2-3 times for enrichment as noted above to derive rVabT⁺ S⁺.This procedure can be done before or after any of the above proceduresrelated to isolating Active(A⁺) or Target recognition positive(T⁺)library members.

If all screens for T, S, and A are accomplished, the final library wouldbe rVabT⁺ A⁺ S⁺ given that there was only one LIGATT and one TARGATTrequired for regulation of the target and thereby represent individualentities which describe target sites suitable for screening for SOMERSwith all three attributes of a CAP. Where there are more than one LIGATTand one TARGATT required for target regulation, i.e., when the target ismultimeric or even monomeric but contains multiple TARGATT domains, thefull CAP, including activity (A+), can only be observed with a bivalentrVab, such as would be found in an active rVab-PEP. In such cases, therVab portion of the active bivalent rVab would not be active on its own.Nevertheless, since it still can identify SOMERS we refer to it as A*.

Clearly, high affinity (less than or equal to about 30 nM) and selectivetarget recognition do not require the antigen pocket of the Vab be madeup of two V domains as found in native Ig molecules but can exist insingle VH domains containing only 3 CDRs. Based on the information inthe art, improvements in making useful single chain (rVvx; i.e., vh orvl) with T⁺, S⁺ and A⁺ properties are expected by utilizing constantdomains other than CH1, i.e., using gamma 2 or 3 or delta. Thisinvention also recognizes the need for solubility of the recombinantproteins used to construct the members of the rVab, rVvx and rVab-PEPlibraries. To be acceptable, changes in solubility would not adverselyeffect VH; VL structure in an rVab.

When using single chain libraries, select the rVvx entities which modifypharmacological target activity via binding to its surface. Refer tothese as the active rVvxT⁺ A⁺ libraries (LIB). Isolate actives based on:

i. those whose binding is modified by the presence of the endogenousligand;

ii. those whose binding is modified by any allosteric regulator of thetarget

iii. those whose binding alters target (i.e. target phosphorylation orassociation with G proteins).

In the case of i and ii, actives are isolated as soluble entities and iniii precipitated by anti PO₄ -protein or G-protein antibodies. In iendogenous ligand is used ad 300× Kd. In all cases harvest positives,amplify, and reisolate.

Group as to common surface domain recognized by rescreening activerVvxT⁺ A⁺. LIB against target in presence of small peptides (10-12 aminoacids) or large peptides made recombinantly (20-50 amino acids) whichdefine the target domain. In this assay, those soluble in presence ofpeptide are grouped together, and all data are used to construct anantibody surface map.

The members of the rVab library which are particularly useful inautomated binding assays and screens for SOMERS at preidentified targetsites possess preferably the following characteristics.

a. ≦30 nM affinity for target;

b. recognized target sites are smaller than those used by endogenousligand signals;

c. possesses agonist or antagonist activity when bound to an activelandscape whether it be those used by endogenous ligand or allostericsites;

d. specificity for binding to only one among many related members of atarget family;

e. little nonspecific binding to unrelated targets and substancesrelated to the assay itself;

f. easy and homogeneous and single tagging with a label;

g. labelling which allows both rapid and sensitive quantitation oftarget binding and;

h. a framework of known structure which delineates the location in spaceof the contact points of the reporter with its target.

The latter attribute is critical to the solution of the 3D structure ofactive SOMERS as it allows the problem of deducing the 3D-shape of theLIGATT on the target surface scanners which are active and in contactwith the target to be solved after obtaining the one dimensional linearamino acid sequence of the reporter with the use of genetic algorithms.The 3D landscape of the LIGATT on the active rVab is directlytransformable into a 3D landscape of the sought after SOMERS.

VI. Identification of Biologically Enhanced Ensemble Pharmacophores(BEEP)

A. Combine structural information from identified members of librarypossessing desired attributes of potency, activity, selectivity, andspecificity

In trying to identify useful rVabs and to deduce the structure of theBEEP, the ability to genetically simplify (e.g., reduction in number orsize) or further diversify (e.g., increased number of randomized aminoacid positions, or increased size) of CDRs and CSRs within active rVablibraries or within one rVab is of critical significance. This isbecause not all contact amino acids contribute the same energy toantibody binding and sometimes one amino acid can account for >99% ofbinding energies. Just the 3 CDRs of one VH can provide 10-100 nM of Igtarget affinity. rVab phage libraries of about 10¹² members withsecondary diversifications in any number of regions can be derived froma small number of active rVabs found initially by processes of theinvention previously described, by PCR as used to construct the rVablibrary (see below) and or oligonucleotide insertion, known to thoseskilled in the art to provide an acceptably large enough source oftarget surface scanners and reporters as envisioned by this embodimentof the invention. In addition, it is clear that active surface scannerrVab will be needed which recognize different local surfaces on thetarget in order to generate sufficiently large amounts of onedimensional amino acid sequence information so as to accurately deduce aBEEP which is not only accurate for predicting the structure of oneSOMER but is capable of predicting the ensemble of active SOMERS whichcan attach to that site.

A particularly novel aspect of this invention is that it establishes away for the CDR regions of a VH or a VL alone or complexed together asrVab to be reduced to a minimum structure which occupies the targetsites recognized by the rVab and have a desirable CAP. An advantage ofidentifying such a minimum structure is the potential reduction oftarget affinity to a level which is competable in standard bindingassays by endogenous ligand and potential SOMERS and of the number ofcritical atoms participating in target contact. The smaller the numberof contact points the simpler the resolution of the BEEP.

B. Create Beeps For Each Active rVab Subset

According to this invention, BEEPS are created which contain thecoordinates and attributes of the active elements of the 3D surface ofactive SOMERS for a particular surface domain on particularpharmacological targets. The starting point for this is groupingtogether of rVabT⁺ S³⁰ A⁺ members of the rVab library according tocommon target surface domain recognized which in the first instance willbe that which is overlapping, or identical to endogenous ligand.

In a preferred embodiment:

a. Each surface group is partitioned and one rVabT⁺ S⁺ A⁺ for that groupis isolated. The VHCH gene is then cloned out and used to derive a newcombinatorial library. To derive this new combinatorial library thecloned rVHCHn is paired with all rVLCL for rVab members which bind tothe common surface.

b. Isolate via panning (as done for the original LIB) all newcombinational rVab members (i.e., rVHCH^(n) : rVLCL^(n) . . . rVab)which are T⁺ S⁺ A⁺ for the original common target surface domain. Thislibrary is called rVab_(VHn). Repeat for each VHCH in the original rVabthereby deriving a rVab_(VHn+1),n+2,n+ . . . set which identifies allrelated VH and VL for a particular surface domain. These libraries willprovide multiple combinations of defined VH genes with all VL's for agiven surface. Alternatively, these various libraries may be made byidentifying specific VL genes and cloning them into libraries containingall VH genes identified for a given surface target.

c. Determine via PCR the amino acid sequence of all VL in the set whichcan bind to all VHs in the library.

d. repeat a-c for all active V_(H) using [V_(L]n),n=1n=2n+ . . . .

e. The spacial coordinates for the framework of the parent antibody inwhich all randomized CDRs were placed, along with the coordinates of thevarious CSR and CDRH3 for the active VH and VL for those entities foundin the particular local target surface domain rVab library under studyalong with the amino acids identified in these CSRs and CDRs are solvedin a genetic algorithm to determine the 3D conformation of thepharmacological target landscape occupied by all active rVab memberswhich recognize the same surface domain. This solution is a biologicalenhanced ensembled pharmacophore (i.e., a BEEP)

f. Repeat for rVab library for other local active target surfacedomains.

g. If any data base is not sufficient, take the relative set of VH genesand excise their CDRH3 domain and replace with a random oligonucleotideencoding a peptide library of preferably 8 to 10 amino acids. Thepotential size of this library is between about 8²⁰ -10²⁰ members.Repeat selections to obtain new diversity enhanced LIB.

C. Use of genetic algorithms to create BEEPS

Creation of the BEEP begins after isolation of a set of active rVabs{Vi}i=N, which contain members (Vi) which have been verified as havingthe desired attributes of affinity, selectivity and activity at thetarget, where N=the number of such members within the set. In thepreferred instance, each active rVab will have all three of the aboveattributes, but it is also possible that only two, or only one, of theattributes will be desired and therefore will be present. For thisdescription, TSA+ will refer to the active rVab irrespective of whichattributes are present. Each TSA+ rVab member is then isolated and itsamino acid sequence determined using procedures known and available tothose skilled in the art. For example, commercially supplied kits and anautomated sequencer (ABI, USA).

According to this model, it is assumed that an active target surfacebinds different rVabs, through the same site of the target surface, andaccordingly, at least a subset of those rVab are expected to possesssimilar surfaces. Thus, finding a recurring, i.e., common, surface motif(which we refer to as the BEEP) in different rVabs indicates either: a)the common rVab surface plays a role in target: rVab interactions; andb) that this interaction could be duplicated by other molecules withsimilar surfaces. Therein, it is a common surface which is responsiblefor the common phenotype of at least a subset of the L_(i) members ofthe original set of TSA+ rVabs. There may be one or more common surfaceswithin the original set of TSA+ rVabs. This duplication takes the formof the BEEP first, and subsequently small organic molecules.

Given such a collection of TSA+ rVabs and their amino acid sequences, apreliminary set of surface scanners {L_(i) }_(i) =N, where each L_(i) isa model of an antibody molecule, is constructed according to theinvention using the canonical structural principals of Chothia (Chothiaand Lesk 1987, Chothia 1989, and Chothia 1992) and the information onthe crystalline form of the parental antibody used as framework forconstruction of the rVab library as described by this invention, N isthe number of such TSA+ rVab surface scanners which define thefundamental geometry which is the position of surface atoms withinacceptable distances from each within a generally known structure. Shapedescriptors rely on known CSR and CDRH3 shapes, and the amino acidsequence within these domains. Subsequently, chemistry characteristics,such as charge, hydrophobic interactions, exposed/buried surface area,hydrogen bond formation etc., known to those skilled in the art will beconsidered.

In the preferred case, each TSA+ rVab contains one VH and one VL chain,with 6 complementary determining regions (CDR) wherein three(CDRVL1,2,3) are within VL and three (CDRH1,2,3) are within VH.Furthermore, in the preferred case, there are the 5, 1 and 6 differentcanonical structures consisting of a different known canonical loopstructure possible for every CDRVL1,2 and 3 respectively, and 3, and 4different canonical structures consisting of known canonical loopstructures possible for every CDRH1 and 2 according to the invention.The CDR for H3, although not canonical, in the parental library willhave one of three defined structures in its parental mode before theamino acids positions within each are randomized. Furthermore, the priorknowledge of rVab framework and relationship of the 6 CDR domains withinthe framework provides additional structural information forconstructing an L_(i) and eventually a BEEP. In addition, as the numberof known antibody structures increases, new canonical structures becomeknown and may be incorporated into the rVab libraries to allow isolationof TSA+ rVabs containing such structural loops.

Each L_(i) can be represented, for the purposes here, by the atomiccoordinates of the constituent atoms of the rVab which is a member ofTSA+ set. The surface (S_(i)) of the preliminary model L_(i) can beparsed by its CSRs and CDRs wherein

S_(i) ≅[(CSR1)_(i), (CSR2)_(i), (CSR3)_(i), (CSR4)_(i), (CSR5)_(i),(CDR6)_(i) ]

wherein 1 through 5 denote CSRVL1, 2, and 3 and CSRH1, 2, and 6 denotesCDRH3, respectively, and wherein with each (CSR)_(i), for L_(i) there isa particular sequence.

The surface (S_(ij)) can be repositioned and reoriented in space bytransforming the atomic coordinates of the Li according to: S_(ij)≅G_(ij) *L_(i), where L_(i), is a model of surface scanner i defined bythe coordinates of its constituent atoms and G_(ij) is a matrix thattransforms L_(i). Furthermore, G_(ij) is parameterized by thetranslation and rotational parameters (Ψ_(i), Xχ_(i), ω_(i), x_(i),y_(i), z_(i))j. Thus, as scanner i is rotated and moved into a newposition j, and the CDR are carried along with it.

The genetic algorithm of this invention, referred to here as DIOGAM,takes the initial set of {L_(i) ⁰ }, where the superscript (⁰) means`preliminary model`, as input data to produce from that data as outputthe theoretical common surface (i.e., the BEEP) which represents thebest overlap in terms of chemistry and geometry for members of the set.

In general, a genetic algorithm (Holland, J. H., 1992 and Goldberg, D.E. 1989, which are herein incorporated by reference) operates on `genes`to produce variation which through selection yields `survivors`. Thegenes of survivors (as judged by `fitness`) are then mutated to producenewer progeny for further fitness selection. Thus, mutated genes,according to the genetic algorithm of the invention DIOGAM, are producedand encode altered surfaces, which in turn are altered phenotypes.

The definition of a "gene" for use in the model of this invention is aspecific sets of values for the parameters of G_(i) : (φ_(i), χ_(i),ω_(i), x_(i), y_(i), z_(i))j. Varying these parameters changes theposition of the surface Sij which we define here as the phenotype of thegiven gene.

Herein, [{G_(i) ⁰ }]j=1,M is a population of M variations of the modelLi, which encompass all possible ways to vary the surface of the model,on each member of the TSA+ rVab set which gives rise to subsequentmodels (1st progeny generation, 2nd progeny generation, nth progenygeneration models [1-n]) wherein n=the number of the generation.

The initial creation of preliminary models follows in one embodiment theComputer Vision algorithm for structural and surface comparison ofproteins (Fisher et al.; 1994) using a small number of points,rotational and translational in nature for unique definition. Thismethod is based on the previous method of the Geometric Hashing Paradigm(Lamdan and Wolfson 1988 and Lamdon Schwartz and Wolfson, 1990). Thismethod finds 3D motifs within different segments or by isolated singleamino acids, independently of any linear sequence of amino acids. Thelater provides for incorporation of all important amino acids or groupsthereof located within the 5 CSRs and 1 CHDH3 and which by themselves donot occur in a singularly linear sequence within any rVab.

Using only distance invariants, this program obtains data from surfacesuperpositioning which is then used to solve for portions of the rVabwhich represent analogous portions of surfaces of ligands directlyinvolved in ligand-target binding requirements, i.e., the `dockingproblem.` Various types of surface superpositioning can be used, andincludes docking of rVabs, one rVab and one target, and one rVab and onetarget related ligand. DIOGAM uses an efficient automated computervision based technique for detection of three dimensional structuralmotifs (Fisher, D., et al., 1992; and Bachar, O., et al. 1993). In thisprocess, seed matches are found first, based on the Geometric HashingParadigm, the clusters of seed matches are found using rotational andtranslation parameters to fix 3D motion. Here the seed matches will bedone within specific sized balls, using different pairs of balls, thesubsequent clustering added by known CSR structure and CSR and CDRrelationships within each rVab. Extensions will be extensive, eventuallyincluding all amino acids within each CSR and CDR, using reiterate evergrowing cycles.

Such clustering and extension (referred to here as additional levelmutations (see below)) can be used for both chemistry and energyanalyses. Modeling will initially be done individually, then in anaggregate manner.

Therein for each progeny generation, the sum of {S_(ij) ^(n) }, whereinj=jth member of the ith scanner as appearing in the nth generation givesus a Target Fitness Landscape (T_(i)): which is a set of numbersrepresenting chemical and geometric properties of the maximallyoverlapped set of S_(ij). For the purposes of this invention, Tn is avector whose components, tj, include but are not limited to scaledelectrostatic energy, buried surface area, hydrogen bonding, and localcurvature.

As the algorithms proceeds, it calculates at each stage, the targetfitness landscape (T) and ascertains a mutational strategy for the nextstage. Thus, depending upon the strategy, all N genes are mutated,producing new phenotypes for which a new value of T is calculated. Theprocess is complete when T can be maximized no further.

Thus DIOGAM alters the set of {φ_(i), χ_(i), ω_(i), x_(i), y_(i), z_(i)} in order to achieve the best overlaps in the general sense (geometry,energy and chemistry) and the result is new Target Fitness Landscapes(i.e., T) defined to be a minimum when maximum generalized overlap hasbeen achieved.

The next or intervening phases of DIOGAM allow variation (i.e.,mutation) in the Li themselves thus the genetic algorithm include sgenetic varation of CSRs and CDRs. For DIOGAM, the mutated gene (i.e.,the augmented or varied gene) is the collection of rotamer angles of theside chains themselves within the CSRs and CDRs. Such changes wouldinclude, as example, changing the rotation around a Cα-Cβ bond(C=carbon), which for a valine put it in result in 3 differentpositions). For an arginine, there are up to 27 rotomers of theguanidium group. In the preferred mode, structural variations will becarried out early on. Considering mutational events, another level ofvariation could be rocking of the models. Further mutation (i.e.,variation) would be changes in the angle between VH and VL from 0-15degrees, which has the effect of shifting the target residues within thegenes over a longer distance which can be considered shifting C αpositions. These mutations will include `catastrophic events` havingglobal implications for the position of the amino acid within the CSR orCDRH3. These mutations enable local minima trapping to be avoided.Although the above mutational events are the first two preferred, theorder of changes will be modified during the overall DIOGAM program.

Note that VH CDRH3 is a special case. This is so because first there areno canonical structures for CDRH3, second, it is by far the largest CDRregion with insertion sizes of up to close to 24 amino acids; and third,because it can influence the angle between VH and VL. Therefore, thisregion is the one of most variations with the least structuralrestrictions.

According to the preferred mode of the invention, there are twopositions within each CSR gene, which do not alter its canonicalstructure, and which are randomized in the rVab lib. as to amino acid.This translates to the possibility of any one of 20 amino acids beingpresent at these two positions within each CSR and CDHR3 within any oneof the Li members selected TSA+ rVab set under analysis. Therein, in thefirst level variation phase of DIOGAM, there is an arbitrary `mutation`,herein meaning rotation, of the gene allowing presentation of thevarious possible rotamers for these two particular amino acids foundwithin one TSA+ rVab at each of the two randomized positions within thegene. Such mutation events will also be used later with VH CDRH3 at itstwo randomized amino acid position.

These mutants will then be analyzed by DIOGAM to derive other sets ofT_(1-n), in the manner described above.

Additional mutational events may also be utilized to produce furtherdiversity to more fully describe the minimum structural requirements todefine the common overlap (i.e., BEEP) which has the best TSA+ phenotypefor the active site of the Target. Mutational events which effectfitness, will involve, but not be restricted to hydrophobic,electrostatic and conformational entropy effects, surface roughness,surface curvature, avoidance of unpaired charges, favorable andunfavorable steric interaction of functional groups and will becharacterized by available programs like COGEN (Bruccoleri, R. E., andKarplus, M., 1987; Novotny, J., Bruccoleri, and R. E. Saul, F. A., 1989;and Tulip, W. R., et al. 1994) and the multiple copy simultaneous searchmethod of CHARMM (Miranker, A., and Karplus, M., 1991; Patai, S. 1989and Brooks, B. R., et al., 1993) using functionality descriptors withfewer atoms (Andrews, P. R., Craik, D. J., and Martin, J. L., 1984) or aspherical approximation to a multi-atom group (Goodford, P. J., 1985 andGoodsell, D. S., and Olson, A. J. 1990) based on time dependent Tartreeapproximation or minimization (Elber, R., and Karplus, M. 1990).

Once these mutational levels (1⁰ -n⁰ level mutations) have been gonethrough one time, for each L_(i) ⁰, there will be new children (perhapshundreds to thousands) of the original parental rVabs. Structuralparameters of the second are then put through the `Nussinov-ComputerVision` algorithm (Fisher, et. al. 1994), which is included herein byreference, to obtain the best alignment. Details of this method and someapplications of the program (Fisher, D., et al., 1992 and Bachar, O. etal. 1993) are included herein by reference. The lowest values of thetarget functions for each Tn, will be different. The values willinclude, but not be restricted to, rms (for geometric overlap), ΔG(Gibbs free energy) and chemistry. The mutational events; will produceprogeny which will be selected as having <rms, <energy and <negativechemistry values than those of the parental targets. Together the sum ofthese values define an overall Target Fitness Landscape for each Tn.

At this stage, DIOGAM will use commercially available algorithms, asdescribed (see Goldberg 1990) by providers, and known to those skilledin the art, to score and register the results of each fitness test. Atthis stage then, there will be a list of (φ_(i), χ_(i), ω_(i), x_(i),y_(i), z_(i) for each L_(i) ^(n) and a running fitness score (Tij^(n)).DIOGAM then goes back to next cycle of genetic variations, doing theseiterations for thousands and thousands of generations, simultaneous, orin an ordered fashion, which at its termination will provide a list ofbest minima, which will be the 1st level BEEP, i.e., the best overlap ofthe surfaces contained within the set of active TSA+rVab.

We have done this manually in the case of two antibodies (NC10 and NC41)to the same site (epitope) on the surface of neuraminidase (Tulip, W.L., et al., 1994) and Malby, R. L., et al., 1994) which have beendefined crystallographically and which provides us with a population,here only containing two members, which approximates the TSA+ rVabpopulation isolated by this invention. Analysis of this population hasshown overlap of antibody CSR and CDR surfaces which are bound to thesame epitope. Therefore, a Sij surface as envisioned by this inventioncan be made.

At this stage, DIOGAM now goes back to the mutation stage and iterates,i.e., arbitrary changes rotamer position, overlapping the set, yet in sodoing producing a slightly different set of φ_(i), χ_(i), ω_(i), x_(i),y_(i), z_(i), but more importantly, finding Ts which are different(higher or lower) from its predecessors. Thus every character of everygene will be updated to reflect the fact that it incrementally(differently) contributed to a more robust phenotype (target fitnesslandscape).

DIOGAM directs the algorithm to enter into its next stage, initiatedafter many such mutational iterations, its crossover or recombinationstage, wherein it creates new combinations of genes, even withoutknowing what is good (better fitness) about an existing gene mutations.These combinations, i.e., mating, of genotypes (or isogenotypes) arebased on T scores, equal phenotype selection of better fitness, whereinfitness is defined as contributing to maximal overall overlap.

It is noted here that overlap is not restricted to physical occupationof identical space, but includes overlap defined, for example, as chargeneutralization wherein, for example, two negative charged residues maybe scored as `overlapping` if they each could be within some distance ofa positive charge.

In this entire process, it is important that the test tube selection ofTSA+ rVab from the large rVab libraries, selects the right combinationof genes which presently in no way can be guessed in advance. Bydefinition, the combination existing in the active TSA+ rVab is`correct` as it contains the surface necessary for desired activityprofile, i.e., consisting of one or more of the desired attributes ofaffinity, selectivity and or activity on the target.

To summarize, in our genetic algorithm, DIOGAM, the gene is the object,the mutation is the change and the early selection is the testing byiteration to get a better number of individual genes. This is thenfollowed by crossover using genetic logic of pieces of genes which areresponsible for the fitness. This crossing over and recombination in thepreferred instance includes deletions and additions of single aminoacids or groups (referred to a seed clustering, or extension orsimplification). With regard to additions, this includes those aminoacids within the CSRs, CDR and framework domains of the rVab which havenot been randomized, and includes those within the CSRs which arecritical to the canonical loop structure itself. The importance ofdeletions and additions to genes as later mutational events is importantas published data (Malby et al. 1994) shows that for two antibodiesbinding to the same antigen epitope, one of the CSR in the pair does notmake contact with the target surface and that large target recognitiondomains may themselves contain much smaller domains which areresponsible for the most of the energy of target interaction (Clacksonand Wells, 1995). For the purpose of this invention, the Ti of the bestcommon overlap, i.e., the BEEP, is related to the existence of a smallsubset of high energy density points in the atoms target surface(Clackson, T. and Wells, J. A. 1995; and Tulip, W. R., et al., 1994),which is considerable less than all contact residues. This is expectedto simplify the alignment (i.e., overlapping) of the L_(i) for exampleif the target domain which is responsible for the TSA+ phenotype of theset selected rVabs is assumed to have just two hot spots then there is avery restricted number of ways a given antibody, known to interact withthe site so as to have a TSA+ phenotype, can bind to that site.

D. Identify small organic molecules active at target sites

1. Use of BEEP as High Volume Screening Reagent

The BEEP provided by this invention may be used as follows to identifySOMERS or drug leads.

a. Use BEEP to electronically screen CHEMFILE to identify SOMERS asdiscovery leads using computer structural programs commerciallyavailable and known to those skilled the art.

b. Use the coordinates of the BEEP to screen via existing computertechnology entire chemical data bases for matching SOMERS.

c. Select a few SOMERS and test in vitro and in vivo to confirmdiscovery lead.

d. Use BEEP to direct synthesis of active SOMERS via techniques known tothose skilled in the art of medicinal synthetic chemistry.

2. Identification of SOMERS using rVAB-Reporters

a. Select: 1-2 representatives of each surface domain group within theactive-selective rVabTSA⁺ library and enzymatically label with, forexample a radionuclide.

b. Establish competition binding assays using endogenous ligand andknown allosteric target regulators as displacer labelled rVabTSAreporter.

c. Screen chemical libraries via standard automated binding assays forSOMERs which displace labelled rVab from its target. Identify all closeanalogs of active SOMERS and perform SAR for target binding.

3. In a preferred embodiment, DISOMERS are identified as follows (SeeFIGS. 21 and 22):

a. Start with all rVab which recognize a surface on pharmacologicaltargets. These can be selected following steps described above.

b. Modify the phage rVab, rVvx library to contain one or two largerandom peptide libraries sufficient to occupy the other one or twoTARGATTS which together make up the active surface of the target. Afteridentifying a scanner rVab to identify one TARGATT identification of theothers is accomplished which may also be done in the presence of thefirst discovered SOMER. Do limited SAR on each SOMER to identify theinactive elements, covalently oligomerize the two or three SOMERS vialinkage through their inactive surfaces to make a DISOMER or TRISOMER.Test in vivo and in vitro to identify best Discovery Lead.

c. Test most potent SOMERs for activity using an in vitro target assay.

d. Test in vitro active SOMERS with best CAP in vivo (via I.P. route toidentify Discovery Leads.

If no analogs exists of originally discovered SOMERS, carry out limitedsynthetic effort, use A*rVabs or rVvx to do a limited SAR binding studyand then select best and test in vitro and in vivo for entire CAP.

If label reporter A*rVab or rVvx for a particular target domain does notuncover SOMERS or none are displayed by endogenous ligand, performsecondary simplication or diversification of CSRs and CDRs, reselect forthe TSA⁺ and carry out 3A again.

Screening for small organic molecular replacements (SOMERS) will be doneby methods known to those skilled in the art using robotic assayemploying labelled n[*]rVab with specific CAP and searching forcompounds which displace [*]rVabT⁺ binding to targets.

e. Excise all rVHCH domains from rVHCHT⁺.LIB, move into the plasmid forbacterial periplasmic expression and create a library of soluble VHCHT⁺.Mix this library of soluble rVHCHT⁺ entities and a phage library ofrVLCL displayed attached to the phage coat protein through its CL region(rVLCL.LIB) to make a combinatorial library wherein only one member ispackaged in the isolated phage and pan against target protein as in 2Aa.After enrichment (2-4 cycles of selection for one or more of the threedesired properties) the genes for the active rVLCL entities areobtained. The genes for the active rVHCHT⁺ entities may then be obtainedin a manner similar to that used to obtain the rVLVL genes. Afterexision of both the rVLCH and rVHCH genes, the Cre-Lox recombinationsystem (see below) may be used to construct a single phage containingboth chains and for expression of the rVab.LIB as a phage displayedfunctional complex. In another embodiment, the libraries may expressedas single chain versions with VH and VL coupled through a linker usingcommercially available kits, such as those from Cambridge according tothe manufacturer. Finally, enrichment and selection of VH:VLcombinations which possess the desired target attribute s may beobtained by, for example, panning.

EXAMPLE 1 Construction of a Recombinant Surface Scanner rVab Library(rVab.lib).

VII. Selection of Parental Fabs of Known Crystalline Structure as rVabLibrary Framework Templates

The amino acid sequences and crystalline structure of the light andheavy chains of the antibody ABXXX which is used as the parental Fab forconstruction of the rVab.library are obtained from the Brookhaven DataBase, the Kabat Data Base, GENEBANK (email: NCBI.NIH.GOV.) or Kabat, E.A., et. al. (Kabat, T. T. Wu et al. 1991). The V regions of the lightand heavy chains are subdivided in domains as follows: the highlyvariable complementary determining regions (CDR), the canonicalstructure region (CSR) within each CDR, and the intervening frameworkregions (FWR) (FIG. 2.5.6). Individual amino acids not within a CSR orCDR, but nevertheless essential to the canonical structure (Chothia andLesk 1987; Chothia, Lesk et al. 1989; Kabat, T. T. Wu et al. 1991;Chothia, Lesk et al. 1992) are also listed (FIGS. 5, 6)

ABxxx is selected as the parental framework template for theconstruction of the ABxxx rVab.lib for recognition of target surfaces byan antibody with a planer type antigen combining site. This selection isbased on the following: 1) availability of the crystal structure of theantibody (bound or free of corresponding binding partner, i.e. antigen);2) the antibody is a member of the planer type combining site group ofFabs (Webster, Henry et al. 1994) which have been found to recognizeprotein surfaces; 3) the antibody has canonical structures for CSR H1-2and L1-3; 4) the antibody's CDRH3 size is in the mid-range of sizes ofCRDH3 (so as to favor equal usage of all 6 CDRs of the rVab in targetrecognition (Wu, Johnson et al. 1993); and 5) the antibody's antigen isa protein (FIG. 3). Parental antibody frameworks found in antibodieswith a cavity and a grove group type combining site (classification asreviewed by Webster [Webster, Henry et al. 1994]) will also be used tomake two additional rVab libraries in a fashion similar to thatdescribed below for the rVab.lib based on ABXXX. Together these threelibraries generate a sufficiently large number of probes for surfacerecognition of relevant binding sites.

In the ABxxx: rVab.lib the natural diversification of antibodies isprovided by placing within the library varied combinations of VH and VLdomains which themselves have varied combinations of the known canonicalCSRs, variable length CDRH3s, and randomized amino acids (one of 20essential amino acids) at one or more amino acid positions within theCSR or CDRs of each V region within each rVab (FIG. 4).

VIII. Creating the Nucleic Acids Encoding the Heavy and Light Chains(rVHCH1 and rVLCL) for ABXXX rVab.lib.

The nucleotide sequence of ABxxx is obtained from Sequences of Proteinsof Immunological Interest, 5th ed. (Kabat, E. A., T. T. Wu et al. 1991);the Kabat Data Base (NCBI.NIH. GOV); or GENBANK. Identification andanalysis of all restriction sites present within these sequences may beaccomplished using a commercially available program (GCG [Univ.Wisconsin, USA], MacVector [IBI, Kodak, New Haven, Conn.], DNAStrider(C. Marck, Gif-Sur-Yvette Cedex, France, Service de Biochemie, Inst.Res. Fundamental, Aloric Energy Commission of France) and SeqEd,[Applied Biosystem]).

Restriction sites endogenous to ABxxx and conflicting with constructionof the rVab.lib as outlined below are removed and replaced with othernucleotides not encoding the conflicting restriction site. This is doneusing sequences which keep unchanged the identity of the parental aminoacid(s).

The sequences are then analyzed again for the changes necessary to placethe convenient and unique restriction sites throughout the V and C genesneeded for library construction as outlined below.

The ABXXX rVab.lib is built according to this invention from separaterVLCL (FIG. 7) and rVHCH1 (FIG. 8) chains which are combined randomly inan in vivo process (FIG. 14). The construction of the rVLCL and rVHCHnucleic acid libraries encoding the rVLCL and rVHCH1 chains, isaccomplished in steps outlined as follows: step 1) oligonucleotidesynthesis: construction of a) amino terminus end (5'V), b) a midregion(MIDV) for VL only, and c) a carboxy-terminus end (3'V) of the V region;step 2) diversification via PCR of some CSRs; step 3) ligation of thesections; step 4) diversification of the remaining CSRs; and step 5)ligation of the appropriate constant (CH1 or CL) region derived by PCRor oligonucleotide construction to generate the complete recombinantheavy and light chain libraries (rVHCH1. lib and rVLCL.lib).

Step 1: Construction of rVLCL.lib (FIG. 7)

In the olignucleotide phase (step A, FIG. 7), construction of a) the 5'(5'VL) end; b) the Mid section (MIDVL) end c) 3' (3'VL) end of the VLregion uses eight synthetic oligonucleotides comprising fourcomplementary pairs. Each oligonucleotide (x) has a complementary matelabelled x'. Two oligonucleotide pairs, a/a' and b/b' are used to makethe 5' end. The MIDVL (c/c'), and the 3'VL (d/d') sections are eachsynthesized from one oligonucleotide pair. The amino acid and nucleicacid positions encoded by the specific oligonucleotides are shown inFIG. 7.

The variance in amino acids at position 2 (within a/a') and 71 (appendedto c/c') necessary to allow for construction of all the desired VL1 CSRsis added during later steps as described below. All oligonucleotides aresynthesized so as to have at least one overlapping complementary stickyend, an absence of hairpin forming ends, and to be noncomplementary tosequences other than that of the desired oligonucleotide joining partnerbased on analysis by a commercially available oligonucleotide primeranalysis software program.

Step 1(a): Construction of 5'VL Section

For construction of the 5'VL end section in step 1(a), theoligonucleotides are first phosphorylated, then mixed together in onereaction mixture, heated, annealed and ligated together using generallyknown molecular biology technology (Sambrook, Fritsch et al. 1990). Theproduct is then isolated and ligated in 60 μl reactions with 1200U T4DNAligase (New England BioLabs) to 5 μg pCLONALL (see FIG. 9 which listsall general use plasmids) digested at restriction site (rs) prs0 and rs4("p" signifies that the location of the restriction site is within theplasmid and outside of the rVab sequence) (Sambrook, Fritsch et al.1990).

DNA is purified from the ligation mixture using Gleneclean II (Bio101),resuspended in water and used for transfection by electroporation(Dower, Miller et al. 1988) of E. coli TG1 (Gibson 1984) grown in brothcontaining 1% glucose for 1h and then plated on dishes in antibioticcontaining media. After overnight (o.n.) incubation at 37° C.,individual colonies are picked. Colonies are identified as rVL3-24.bactfirst by diagnostic PCR using primers pCFWD and pCBCK (see Primer Table,FIG. 10) and subsequently confirmed by sequence analysis via automatedan ABI sequencer and commercially available related kits as outlined bymanufacturer (ABI,USA). Storage of positive clones at -70° C. is done inbroth (Miller, 1972) containing 15% (v/v) glycerol.

Step 2: Diversification by PCR

Toothpicked frozen glycerol stocks of rVL3-24 are used in PCR reactionsto append primers conferring diversification to the rVL section. One ofthe five different CSRL1 diversified with random amino acids at twopositions is used as the FWD primer at the 3' end of the parentalab/a'b' 5'VL section. The BCK primer for the 5' end comprises nucleicacids encoding one of the three different amino acids I, V or S atposition VL2, and the amino acid of the parental ABXXX at position VL1.These appendings are done in 5 primary PCR reactions, each containingone FWD primer (i.e., L1.1FWD, L1.2FWD, L1.3FWD, L1.4FWD) or L1.5FWD)and one of three different BCK primers in the following combinations:L1.1-3BCK primer mixed with the 3 reactions containing L1.1, L.12 andL.13 FWD primers, and L1.4BCk and L1.5BCk mixed correspondingly with oneof the two remaining L1-FWD primers. Subsequently, amino acids VL34-44are appended to the primary PCR products in secondary PCR reactions bytaking an aliquot of the primary reaction and carrying out secondary PCRwith primers L1ALLFWD and L1ALLBCK. The products of the secondaryreactions are kept separate and are labelled rVL1-44CSR1.1-5.lib.pcr.These constructs allow subsequent generation of all 5 known canonicalCSR L1 in the rVL.lib after cloning when these products are joined withthe appropriate MIDVL section having one of three different amino acidsin position VL71. Each of the primary PCR uses Taq polymerase, FWD andBCK primers as noted above, in 50 μl reaction mixtures and is cycled 25times (94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min). Thesecondary PCR reactions (25 μl) use fresh Taq polymerase and 1 μl ofamplified appended diversified primary PCR reaction mixture product, FWDand BCK primer pairs as noted, and the reaction is cycled 30 times (94°C. for 1 min, 55° C. for 1 min and 72° C. for 2 min). A list of thesequences of all primers appears in Primer Table (FIG. 10).

In step C, the five products of the secondary amplification reaction ofcorrect size, are designated rVL1-44CSR1.1-5, and are isolated on lowpercentage acrylamide gels, recovered, restricted and ligated topCLONALL precut with prs4 and rs2 and cloned via electroporation (Dower,Miller et al. 1988) into E. coli as described (step B, FIG. 7). Thesefive 5'VL section products are designated rVL1-44CSR1.1-5.lib.bact.Twenty clones of each library are checked first by diagnostic PCR andsubsequently five (5) clones are analyzed for diversification of CSR1 byautomated sequencing as described above using pCFWD and pCBCK sequencingprimers and commercially available kits (ABI,USA). This proceduregenerates greater than 10⁴ transformants per each of the five VL1 CSRs.

Step 1(b): Construction of the MIDVL Section

In parallel fashion, a second set of reaction steps A-C constructs theMIDVL section of rVLlib. The MIDVL section originally contains aminoacids rVL53-68. The oligonucleotides for this reaction are contained inthe one pair c/c'.

In step A, each oligonucleotide is phosphorylated, the pair hybridizedtogether under annealing conditions, and the c/c' double stranded DNAcomplex is purified and ligated in a 60 μl volume with 1200U of T4DNAligase (New England BioLab) to approximately 5 μg rs2 and prs5 cutpCLONALL (Sambrook, Fritsch et al. 1990). Ligated product is isolatedfrom the mixture using Genecleanb II (Bio101), resuspended in water andused to transform E. coli via electroporation (Dower, Miller et al.1988). After 1 hr in broth containing 1% glucose, the cells are placedon dishes in antibiotic containing media. After overnight incubation at37° C., individual colonies are picked and the MIDVL sectiontransformants are identified from among 30 transformants generated bydiagnostic PCR. Confirmation of sequences is by automated sequencingusing an ABI automated sequencer using pCFWD and pCBCK primers(ABI,USA). Positives are labelled rVL53-68.bact. and frozen glycerolstocks are produced.

In step B diversification, PCR is used to append diversified CSRL2 tothe 5' end of MIDVL. Three different amino acids at VL71 (i.e., Y, F andA) followed by restriction site rsC between VL72 and VL76 followed by ars4 restriction site are appended with primers to the 3' end of MIDVL.These additions are done in three separate reaction mixtures, one eachcontaining FWD primer L2.71YFWD, L2.71FWD and L2.71FWD. All three FWDprimers contain the rsC site which will allow joining of MIDVL to 5'VLsections. For each of these reactions, the BCK primer is L2ALLBCK whichcontains an rsB site as well as DCSRL2 diversified at amino acid VL50and 51. Each mixture contains a toothpicked frozed glycerol stock ofrVL53-68 (see Primer Table, FIG. 10 ), Taq polymerase, in 50 μlmixtures, and is cycled 25 times (94° C. 1 min, 60° C. 1 min 72° C. 2min).

In the following step C, approximately 1 μg of the amplified diversifiedappended MIDVL products are isolated using Magic PCR Preps (Promega),cut with prs1 and rs4, reisolated and ligated to 5 μg pCLONALL precutwith prs1 and rs4 in 60 μl volume with 1200U T4DNA ligase (New EnglandBiolabs) (Sambrook, Fritsch et al. 1990). The ligated plasmid DNAproducts are isolated using Geneclean II (Bio101), resuspended in waterand used to electroporate E. coli to generate, as noted above, a libraryof transformants (Dower, Miller et al. 1988). The three separate groupsof successful transformants (one for each type of VL71) are identifiedby diagnostic PCR and confirmed regarding diversification of VLCSR2 byautomated sequencing of 10 clones of each group. These transformants aredesignated rVL 38-73CSR2:71 (Y,F,A) lib.bact. This procedure gives ≧10⁴transformations for each group.

Step 1(c): Construction of the 3'VL Section of rVL

In the third set of parallel steps A-C, the 3'VL section of rVL.lib isconstructed. This section is originally built to contain amino acidsVL72-90 and uses the one oligonucleotides pair d/d'. In step A, thispair is phosphorylated and the two oligonucleotides annealed. The doublestranded complex is then isolated and is ligated to pCLONALL precut withprs0 and rs4'. Ligated product is isolated and used to transform E. colivia electroporation (Dower, Miller et al. 1988) as above. 3'VL sectiontransformants are isolated from among the transformants generated, anddiagnostic PCR is preformed on twenty of them, the positives beingconfirmed by automated sequencing and labelled rVL76-90.bact. Frozenglycerol stocks are prepared.

In the next phase, diversification (step B), the six diversified CSRL3s,followed by a new prs5 site, as well as amino acids VL72-75 whichcontain the convenient restriction site (rsC), are appended to VL76-90to make the following 5'VL PCR product: rVL72-100CSR3.1-6.pcr.Diversification of CSR3.1-6 occurs at positions VL92 and 93. Theseprocesses are done in six (6) separate 50 μl PCR reactions eachcontaining one L3.1-6FWD primer, all containing L3ALLBCK (see PrimerTable, FIG. 10), and Taq polymerase in 50 μl mixtures. The reactions arecycled 25 times (94° C. 1 min, 60° C. 1 min and 72° C. 2 min).

In step C, the amplified diversified appended products are isolatedusing Magic PCR Preps (Promega), cut with prs2 and rs5, reisolated andligated into pCLONALL precut with prs1 and prs5. The ligated plasmid DNAproducts are isolated and used to electroporate E. coli to generate alibrary of transformants as noted above and designatedrVL72-100CSR3.1-6.lib.bact. This procedure gives greater than 10⁴transformations which are identified by diagnostic PCR and sequencing tocontain appropriately randomized amino acids at the diversifiedpositions within VLCD3 for each of the six (6) VLCSR3s.

Step 3: Ligation

In step 3, the 5'VL and MIDVL sections are joined (see FIG. 7). Five μgof DNA of each of the five rVL1-44.libs (i.e., CSR1.1-5) is digestedwith rsB and rs5 and ligated to 1 μg of insert isolated from the threerVL38-70CDRL2:71* using 1200U T4DNA ligase ((New England BioLabs)(Sambrook, Fritsch et al. 1990). In these reactions, ligation pairing of5'VL[rVL1-44CSRs] to MIDVL[rVL38-76CSR2:71*] is maintained as:5'VL1.1-3×MIDVL2:71Y; 5'VL1.4×MIDVL2:71F and 5'VL1.5×MIDVL2:71A tocreate the five rVL1-76CSRD1&2.DNAs. Each of these is used toelectroporate E. coli (Dower, Miller et al. 1988).

The bacteria are then grown in broth containing 1% glucose for 1 h andare plated on dishes in antibiotic containing media, After overnightincubation at 37° C., individual colonies are picked and arecharacterized first by diagnostic PCR and then by automated sequencing.Some 100 colonies are analyzed by diagnostic PCR and 20-30 by sequencingto confirm the random presence of different CSR pairing and diversifiedamino acids within the various CSRs. Frozen stocks of the five groupsare then prepared and are designated rVL1-76CSR12.lib.bact.

In step F, the extended 5'VL halves, consisting of the fiverVL1-76CSR1&2.libs., are joined in 30 separate PCR reactions incombinatorial fashion with the six 3'VL halve sections, consisting ofthe six (6) rVL72-100CSR3.1-6.lib. This process generates 30 full lengthrVL1-100CSR1&2&3.lib. (as diagramed in FIG. 7). In each of these libraryconstructions, about 5 μg of DNA of each of the five rVL1-71CSR1&2.libs(i.e., CSR1.1-5) is digested with rsC and prs5 and ligated to 1 μg ofeach of the inserts isolated from the six rVL72-100CSR3.1-6 digestedwith rsC and prs5 using 1200U T4DNA ligase (New England BioLabs)(Sambrook, Fritsch et al. 1990) to create the 30 rVL1-100CSRD1&2&3.dnapreparations. Equal aliqouts from each ligation mixture are pooled andthe pooled DNA is purified using Geneclean II (Bio101) and resuspendedin 30 μl water to create the completed rVLCL.lib.dna. PCR is then usedto append to the 3' end of this DNA library, the nucleotides encodingthe remaining amino acids of VL (i.e. rVL101-107), amino acids at the 5'end of CL (i.e., amino acids CL108-110), and within this sequence theconvenient rs3 site. The rs3 site, also designated the rsCLLNK site(FIG. 9), subsequently allows the joining of rVL.lib with its cloned rCLsection.

These appending reactions are done by carrying out a PCR reaction withan aliquot of the purified rVL1-100CSR1&2&3.lib.dna, the primersLJCLLNKFWK and L1ALLBCK, and the Taq polymerase in 50 μl volume mixturescycles. The PCR reaction is cycled 25 times (94° C. for 1 min, 60° C. 1min and 72° C. for 2 min).

Amplified DNA is then purified using Magic PCR Preps (Promega). Aftersuspension in water, 1 μl g of the purified DNA is digested with rs2 andprs5 and ligated to 5 μg of pCLONALL DNA precut with rs2 and prs5 using1200U T4 ligase (Sambrook, Fritsch et al. 1990) and used toelectroporate E. coli (Dower, Miller et al. 1988). The bacteria grown inbroth containing 1% glucose for 1 h are then plated on dishes inantibiotic containing media. After overnight incubation at 37° C.,individual colonies are picked and characterized first by diagnostic PCRand then by automated sequencing. Some 100 colonies are examined bydiagnostic PCR and some (about 5-10) by sequencing to confirm thepresence of amino acids VL1-110 and the random presence of different CSRpairings and diversification of amino acids within the various CSRs.More than 10⁸ transformants are generated in this process and a frozenstock of the library is then prepared and designated rVL.lib.bact.

In the last step (step G) of rVL.lib construction, DNA from rVLlib isdigested with rs2 and rsJCLNK, and 1 μg is ligated to 5 μl g ofpVLACCEPTOR (FIG. 9), precut with rs2 and rsJCLLNK, using 1200U T4ligase (Sambrook, Fritsch et al. 1990). The product is then purifiedfrom the ligation mixture using Gleneclean II (Bio101) and resuspendedin water. This material is used to electroporate E. coli (Dower, Milleret al. 1988), and the bacteria are grown, after1 hr in brothsupplemented with 1% glucose, overnight at 37° C. on dishes inantibiotic containing media. Individual colonies are picked andcharacterized by diagnostic PCR and automated sequencing to confirm thepresence of CL in the library. Frozen glycerol stocks ofrVL1-110ΔCSR1-3lib are made and designated rVLCL.lib.bact (FIG. 7).

The above detailed reactions where double amino acid randomizationoccurs within each CSR theoretically allows the construction of 2000,400 and 2400 different CSR L1,2,3 respectively, and a rVLlib size of1.92×10⁹. This exceeds the largest published recombinant VL library madeby similar (Griffiths, Williams et. al. 1994) technology by about 2fold.

IX. Construction of the Constant Regions of ABxxx

The constant region (C) of the light (CL) and heavy chain (CH1) regionfor the selected parental Fab ABxxx (FIG. 9) is obtained either byannealing and ligating a series of synthetic overlappingoligonucleotides, as done for the V regions, or via standard PCR of theC regions of ABxxx or any other antibody mRNA or DNA with identical Cregions. Nucleic acids encoding specific antibodies may be obtained fromhybridmas from various sources including the ATCC. In either case, theconstructions includes the removal of endogenous restriction sites thatinterfere with library construction and the creation of a number ofconvenient restriction sites at and around the 5' and 3' ends of the Cregions so as to allow simple cloning into pCLONAL, pEXPRESSION and pV(Hor L)ACCEPTOR (FIG. 9). For both CH1 and CL regions, the C genes haveinserted within them an rs3 site for specific joining of V and Csections of rVL at or about the natural V/J gene junction for heavy andlight chains. These sites are referred to as either rsJCHLNK andrsJCLLNK respectively. In constructing the C sections, these twojunctional rs are appended by standard PCR using BCK primers CLBCK andCHBCK and FWD primers CLFWD and CHFWD (see Primer Table for sequencedetails (FIG. 10).

The parental C nucleic acid sequence of ABXXX is amplified by PCR withTaq polymerase using primers CLFWD and CLBCK which places the rs3restriction site within the JC segment of the parental Fab at the 5' endof the C sequence and two stop codons (TAA) and the rs4' site (AscI)just outside the 3'-end of the C region. The reaction mixture (50 μl) iscycled 25 times (94° C. for 1 min, 60° C. for 1 min, and 72° C. for 1min.) and the amplified appended C sequence is purified using Magic PCRPreps (Promega) and resuspended in 50 μl water.

The reaction amplifying the parental Fab CH1 gene of ABXXX is identical,except for the following: the primers for the PCR reaction aredifferent, being CHFWD and JCHBCK, and the CHFWD primer contains a Not1site at the 3' terminus of the CH1 region.

To complete construction of the VLCL, the amplified and J appendedrecombinant VL diversified CSR1 and 2 and 3 (rVLCSR1&2&3) genes arejoined to the amplified CL gene in the standard ligation fashion usedabove, or using PCR (Horton, Hunt et al. 1989). Assembly PCR reactions(25 μl) use Taq polymerase, 1 μl amplified parental JC, and 0.8 μl ofthe rVL.lib gene from above. The appropriate VLBCK primer is usedtogether with the CLFWD and the reaction cycled 30 times (94° C. for 1min, 60° C. for 1 min. and 72° C. for 2 min.).

X. CONSTRUCTION OF rVHCH1.lib (FIG. 8)

In the oligonucleotide phase (step A), construction of a 5' and 3' halfof the VH region is accomplished using 16 synthetic oligonucleotides,comprising 8 complementary pairs. Six oligonucleotides are for the 5'half and are labelled VH a-c with their complementary partners labelledVH a'-c'. Within the 5'VH half, the oligonucleotide b/b' pair has thersB restriction site between amino acids rVH 22-26. Ten oligonucleotidesare for the 3' half and are labelled VH d-f and d'-f'. Construction ofthe 3' half of the VH region is done in a similar fashion but uses threeforms of the "e" complementary pair, designated as follows VH e/e', VHe2/e2' and VH e3/e3'. These correspond to the "e" oligonucleotides witheither a valine (V), alanine (A) or arginine (R) at amino acid positionVH71, respectively.

In the annealing step, three types of the 3'VH half are constructed:3'VHdef/d'e'f', 3'VHde2f/d'e2'f' and 3'VHde3f/d'e3'f. The variance in"e" oligonucleotides within the 3'VH half is necessary to allow forsubsequent construction in the rVHlib of all four of the known CSRH2 asoutlined below. All oligonucleotides are synthesized so as to have aleast one overlapping complementary sticky end, an absence of hairpinforming ends, and an absence of complementary sequences other than thoseof the desired oligonucleotide joining partner based on analysis by acommercially available oligonucleotide primer analysis software.

Construction of 5' half of the VH Region

For constructing the 5' half of the VH region, the appropriateoligonucleotides are phosphorylated and are mixed together in onereaction mixture, after which they are heated and are annealed andligated together using generally known molecular biology technology(Sambrook, Fritsch et al. 1990). As outlined, the first phase annealingand ligation (step A, FIG. 8) allows the formation of the 5' VHabc/a'b'c' pair. In the next step (step B), the correct construct of 5'VH, containing a convenient rsB within its b/b' segment, is amplifiedwith primers 5'VHFWD and 5'VHBCK (a list of names and sequences forprimers used in VHCH1.lib construction appears in the Primer Table, FIG.10) by carrying out PCR on an aliquot of the ligated and isolated abcDNA duplex product of step A. In this step, an aliquot from the step Areaction is amplified using the above noted primers and Taq polymerasein 50 μl reactions; and is cycled 25 times (94° C. 1 min, 60° C. for 1min, 72° C. for 2 min.). The amplified DNA is purified using Magic PCRPreps (Promega) and is suspended in 5 μl water.

Next, the product of the amplification reaction having the correct sizeand designated rVH1-51, is cut at rs4 (Not1) and prs1. The cut fragmentis purified by Magic PCR Preps (Promega) and 1 μg is ligated in a 60 μlvolume with 1200U of T4 DNA ligase (New England BioLabs) to 5 μg of rs4and prs1 digested pCLONALL (Sambrook, Fritsch et al. 1990). DNA ispurified from the ligation mixture using Geneclean II (Bio101)resuspended in 30 μl water and electroporated (Dower, Miller et al.1988) into E. coli which is then grown in broth containing 1% glucosefor 1 h and plated into antibiotic containing media. After overnightincubation at 37° C., individual colonies are picked and identified.Transformants containing the recombinant parental 5'VH half, rVH1-51,are identified by diagnostic PCR for appropriate size (with plasmidprimers pCFWD and pCBCK). Those transformants suspected of containingthe rVH1-51 are expanded. The nucleic acid amplified with PCR usingPCFWD and pCBCK are sequenced via automated ABI sequencing withcommercially available kits as outlined by the manufacturer (ABI,USA) toconfirm the identity of the rVH1-51 fragment. Cultures are then grownand stored as frozen glycerol (15% v/v) stocks and designatedrVH1-51bact.

In the next step, step C (FIG. 8), a diversified version of each of thefour known CSRH2 is appended to rVH1-51. This process is done in fourseparate standard PCR reaction mixtures (see above). Each reactionmixture comprises the rVH1-51 fragment (obtained from toothpicked frozenglycerol bacterial stocks), one of four FWD primers (H2.1FWD, H2.2FWD,H2.3FWD and H2.4FWD) and the BCK primer H2ALLBCK. The four FWD primersare constructed to span from amino acid 47 through 59 of CSR2₁₋₄ andcontain amino acid diversification at position 53. The four libraryproducts, are isolated, and are cut at rsB and prs5, and then 1 μg ofeach purified DNA product is ligated using T4DNA ligase to 5 μg pCLONALLprecut at rsB and prs5.

As described above, the ligated DNA is purified and used in step D totransform E. coli via electroporation. Transformants are isolated andcharacterized first by diagnostic PCR and then by automated sequencingto contain appropriate examples of the randomized diversified versionsof all four CSRH1. Frozen stocks of each, designated rVHrsB-59CSR2.1-4lib. bact. are made.

Construction of the 3' Half of the VH Region

In a parallel fashion, another set of reaction steps A-C are conductedto construct the 3' half of the VH region which incorporates nucleicacid encoding amino acids 57-95 of the variable heavy (VH) chain (FIG.8). The oligonucleotides for this reaction contain the three sets ofpairs of VH oligonucleotides, e/e' and e2/e2' and e3/e3' in which aminoacid VH71 is valine, alanine or arginine respectively. Appropriatemixing (as outlined above) allows for annealing and ligation of thethree different rVH57-95 double stranded complementary oligonucleotides3'VHdef/def (i.e., VH57-95[71V]) and 3'VHde2f/d'e2'f (i.e.,VH57-95[71A]) and 3'VHde3f/d'e3'f (i.e., VH57-95[71R]). Aliquots ofthese three reactions are then amplified and appended with rsD and prs5sites in step B by PCR using 3'VHFWD and3'VHBCK. These reactions containTaq polymerase, as described above, and are cycled 25 times (94° C. for1 min, 60° C. for 1 min, 72° C. for 2 min). The correct products arepurified using Magic PCR Preps (Promega), suspended in 50 μl water andare then cut at prs2 and prs5 and reisolated. Approximately 1 μg of thereisolated rVH56-95 gene fragment is ligated into 5 μg pCLONALL precutwith prs1 and prs5. Plasmid pCLONALL with the rVH56-95 insert isisolated and purified using Geneclean II (Bio101), and is used in step Cto transform E. coli by electroporation (Dower, Miller et al. 1988).Transformants are selected, and the correct three products,rVH56-95:71V;A;R, are identified by diagnostic PCR and confirmed byautomated ABI sequencing. Frozen stocks of each, designatedrVH56-95[71V;A; R].bact. are made.

Completion of construction of the nucleic acids encoding the four knownCSRH2 regions genes is accomplished in steps D and E. The threerVHrsD-56-71*-95-prs5 inserts, freed by digestion of plasmid DNA areligated to the four rVHrsB-59CSR2.1-4.lib which have been precut at rsDand prs5. The resultant rVHrsB-95CSR2.1-4 library is cloned into E. coliusing the standard purification, ligation and electroporation processesoutlined above. Transformants are isolated and about 50 arecharacterized by diagnostic PCR and 20 by automated sequencing toconfirm that they contain the expected diversified versions of the fourknown CSRH. The ligation combinations of rVHrsB-59 CSR2 and rVH56-71*-95necessary to construct the fully diversified rVHCSR2 library arerVHrsB-59CSR2.1lib. with rVH56-95:71V; rVHrsB-59CSR2.lib. withrVH56-95:71A; and rVHrsB-59CSR2.3 and 2.4lib. with rVH56-95:71R in stepsD and E.

Step F, comprises sequential PCR reactions to append to the 3' end ofthe four diversified CSRH2 constructs rVHRSB-95CSR2.1-4 diversifiedCDRH3 s of different lengths and the convenient JCH1LNK restrictionsites (i.e., rs3), and at their 5' ends diversify their parental CSRH1and to append nucleic acids encoding VH17-rsB-24. The final PCR productsof these reactions are designated rVH17-118CSR1&2&3.lib and contain allcombinations of the diversified known CSRH1&2's and diversified CDRH3 ofthree different lengths.

These steps are carried out in the following 36 PCR reactions. Ninealiquots of each of the four different toothpicked frozen glycerolstocks of rVHrsB-95ΔCSR2.1-4lib.bact. are added to separate 50 μlprimary PCR reaction mixtures containing Taq polymerase. The forwardprimers H3.5FWD, H3.7FWD and H3.10FWD are added to 3 of the 9 tubescontaining each of the four CSR2s.bact. To each triplicate set of uniqueforward primers is added one of the following: the BCK primers H1.1BCK,H1.2BCK, or H1.3BCK. These primary PCR reactions are cycled 25 times(94° C. for 1 min., 60° C. for 1 min. and 72° C. for 2 min.). Followingcompletion of the primary PCR, aliquots of each of the 36 reactions aretaken for a secondary PCR reaction with new Taq polymerase, and primersH31FWD and H31BCK. The secondary reactions append VH100-rs3-118-rs4 andVH17-rsB-24 to the 3' and 5' ends respectively. The products aredesignated rVH17-118CSR123.lib. followed by a combination number (e.g.,1.1×2.2 ×3.5) which denotes the combinatorial arrangement of the threeCDRHs in these products. Each of the 36 library products arecharacterized by diagnostic PCR and sequence analysis. Aliquots of the36 libraries are pooled to generate the rVH17-118CSR1&2&3.lib.

In step G, DNA from the rVH17-118CSR1&2&3 library is digested with rsBand rs4. The digested DNA is purified using Magic PCR Prep (Promega)ligated into pCLONAL cut with rsB and rs4, purified and used totransform E. coli as detailed above. The transformants are isolated,characterized and designated rVHrsB-118CSR1&2&3.lib.bact.

In step H the rVHrsB-rs3 inserts are removed from the DNA of therVHrsB-118&2&3.lib using restriction enzymes specific for rsB and rs3 toform fragments designated rVHrsB-114CDR1&2&31.3. These fragments areligated using T4DNA ligase (New England BioLabs) to 5 μg rsB and rs3digested rVH1-51-rs3.bact. DNA. The product is then isolated, purifiedand used to transform E. coli to generate rVH1-JCHLNK-ΔCSR1&2&3lib.bact.Individual clones from the library are then isolated and their sequenceis confirmed by diagnostic PCR and sequencing. The library is thenstored as frozen glycerol stocks. The bacterial transformants containingthis library contain the canonical CSRH1 and H2 regions diversified ingreater than one amino acid position, and CDRH3 of three differentlengths and diversified in greater than one amino acid position. Thisprocedure gives at least 10⁵ transformations which are identified bydiagnostic PCR and sequencing to contain appropriately randomized aminoacids at the diversified positions within the CSRH2 and H3 regions forthe rVH1-114CDR2-3.library.

In step I, 5 μg of the rs2 and rs3 precut pVHACCEPTOR DNA (also referredto as pVH-CH, FIG. 9) is ligated to the rs2 and rs3 released insertrVH1-JCHLNKDCSR1&2&3.lib DNA (also referred to as rVHlib, FIG. 8), andthe recovered purified product is designated rVHCH1.lib. This rVHCH1.libproduct is used to transform E. coli to generate a frozen stock ofbacteria containing the rVHCH1.lib. Greater than 10⁶ total members areobtained.

XI. VH and VL Library Sizes:

The above detailed reactions where two amino acid randomizations occurwithin each CSR theoretically allows the construction of 1200, 1600 and1200 different CSR H1,2,3 respectively, and a rVH library size of2.3×10⁹. This exceeds the largest published recombinant VHCH1 librarymade by Similar technology (Griffiths, Williams et. al., 1994) by onlyabout 2 fold. A smaller rVH library can be made using only 2randomizations within the CSRH1 and H2 and one randomization within eachof the three differently sized CDRH3. This procedure theoreticallyallows the construction of 1200, 1600 and 60 different CSR H1,2,3respectively, and a rVH library size of 1.152×10⁸. This is similar tothe largest rVHCH1 library reported. The procedure outlined below allowssubsequent pairing of individual members of such sized rVHCH1 librarieswith individual members of equally sized rVLCL libraries (i.e., of 10⁹as noted above and FIG. 4) on one piece of DNA in single bacteria. Basedon the sizes of the rVHCH1 library and rVLCL library that are generatedabove, the potential size of the combinatorial rVab.lib (i.e.,VHCH1lib×VLCL lib) is greater than 10¹⁸ members (FIG. 4).

XII. Construction of the rVab.lib (the VHCHllib×VLCLlib Combinatoriallib.) (FIGS. 11,12,14)

In this section the phagmid (fd.o slashed.) which carry the rVLCLlib,designated Lox Receiver (LoxREC) (fd.o slashed.RECEIVER, FIG. 11) andthe plasmid (p) which carries the rVHCH library, designated Lox Provider(LoxPro) (pUC19PROVIDER, FIG. 11) are constructed and then are randomlyrecombined in vivo within individual bacteria onto a single phage vector(fd.o slashed.CARRIER) which expresses the rVab rCHCH1 and rVLCL genesand produces on the surface of the phage functional versions of the rVabrVHCL1:rVLCL proteins. The rVab library construction phase is outlinedin FIGS. 11, 12.

Construction is begun by reamplification of the rVHCH1 librarymaintained in the pVHACCEPTOR.lib.bact. using PCR, as described above,with primers pCFWD and pCBCK. The DNA product is isolated and cut withVHrs2' (Nco1) and VHrs4 (Not1) and is ligated using T4 ligase andstandard methodology into LoxPRO precut with Nco1 and Not1. The LoxPROused in this example is fashioned after the pUC based plasmid asdescribed by Griffiths, A. D. et al. 1994) and contains an endogenousCH1, bounded by a Sfil and Not1 rs, preceded by a ribosome binding site(rbs), an in frame LpelB leader sequence (LpelB), followed by an inframewild type loxP sequence (Hoess et al. 1982) and then an inframe mycsequence. In LoxPRO upstream from the LpelB is a mutant loxP511sequence. DNA from the ligation mixture is purified and electroporated(Dower, Miller et al. 1988) into E. coli TG1 (Gibson 1984) to create thepUC based library LoxPRO.rVHCH1lib. (i.e., pUCLoxPROVIDER-rVHCH1lib).More than 10⁸ clones are obtained and the diversity is confirmed bysequencing independent clones.

In parallel, DNA is purified from the rVLCLlib.bact. (FIG. 8) andamplified by PCR with primers pCFWD and pCBCK. The PCR product isisolated, cut with VLrs2 (ApaL1) and VLrs4' (Asc1) and ligated usingstandard methodology into fd based LoxREC (i.e., fd.oslashed.DOGRECEIVER). In LoxREC, upstream from the endogenous VHCH gene,and to be replaced by the incoming rLVCL.lib. there is an endogenous CLgene which is preceded by a leader sequence which ends in a ApaL1 inframe sequence which is followed by two terminator triplet codons. Theendogenous CL gene is followed by two terminator triplet codons, an Asc1and HindIII restriction site, and a mutant 511 loxP site (Hoess et al.1986). DNA amplified by PCR is purified using Magic PCR Prep. The DNA isthen cut with ApaLI and AscI and the digested DNA (about 6 μg), ispurified on a 1.5% low melting-point agarose gel using Magic PCR Prep(Promega). Approximately 1 μg of the purified and cut rVLCL.lib DNA(FIG. 7) is ligated to about 5 μg of digested fdDOG-2loxVkdel (Sambrook,Fritsch et al. 1990) in a 60 μl volume with 1200U of T4 DNA ligase (NewEngland Biolabs) (FIG. 11). Ligated DNA is purified from the ligationmixture using Geneclean II (Bio101), resuspended in 30 μl water andelectroporated (Dower, Miller et al. 1988) into four 50 μl aliquots ofE. coli TG1 cells grown in 1 ml 2×TY broth containing 1% glucose for 1h.Cells are then plated in dishes (Nunc) in TYE (Miller, 1972) medium with12.5 μg/ml tetracycline (TYE-TET). After overnight incubation at 37° C.,colonies are scraped off the plates into 7 ml 2×TY broth (Miller, 1972)containing 15% (v/v) glycerol for storage at -70° C.

The frequency of inserts is determined by PCR for each of the pools.Sequence diversity is confirmed by sequencing 8 clones of each pool. Thepools are then combined to create the rVLCL.lib in fd.o slashed.DOG asrVdLlib. outlined above. DNA from the ligation mixture is purified andelectroporated (Dower, Miller et al. 1988) into E. coli TG1 (Gibson,1984) to create the library rVLCL.lib in fd.o slashed.DOG having greaterthan 5×10⁸ clones. Diversity is confirmed by sequencing 30 independentclones.

Step 4: In vivo Recombination of VHCH1 and VLCL Genes

In this step, summarized in FIG. 14, VHCH1 and VLCL genes are recombinedin pairs, onto single pieces of DNA to make the rVab library. Individualmembers of the VLCL and rVHCH1library are placed within a singlebacteria via sequential incorporation within that bacteria of the rVLCLmember via phage mediated infection and of the rVHCH1 member viaDNA-mediated plasmid transformation. Once inside the bacteria, the twochains are combined onto the same piece of replicating DNA (fd.oslashed.CARRIER) within the bacterium by the P1 CRE recombinase,provided by P1 phage infection, which catalyzes recombination at loxPsite in a process termed `recombinatorial infection` (Waterhouse,Griffiths et al. 1993). The process of recombinatorial infection forexpressing recombinant proteins was originally described by Sternbergand Hamilton (Sternberg and Hamilton 1981); and Hoess et al. (Hoess,Ziese et al. 1982; Hoess, Wierzbicki et al. 1986) which are incorporatedherein by reference and depicted in FIG. 14. In the process according tothe invention, only those bacteria transformed with a rVHCH1/rVLCLcombination (i.e., an rVab member) survive. Given the size of therVHCH1library (greater than 10⁸, see above) and the rVLCL library(greater than 10⁸, see above), this type of combination, given unlimitedbacteria, could yield a rVab.lib of greater than 10¹⁷ members.

According to the invention, the diversified rVLCL.lib is cloned into atetracycline^(R) fd phage (1st antibiotic resistance) containing any VHchain which is easily recognized and which will be replaced later in theprocess by rVH.lib chains. The diversified rVHCH1 chains are cloned intoprovider ampicillin resistant plasmids (2nd antibiotic resistance). Thetwo libraries are then joined in E. coli via phage infection with fdphage containing the receiver VLCL chains (the rVLCL.lib) of bacteriapreviously transformed with plasmid DNA containing provider VHCH1chains. A 1 liter culture of these bacteria is then co-infected with fP1which is chorampenicol resistant (3rd antibiotic resistance) carryingthe Cre recombinase. fd phage recovered from expanded colonies resistantto the antibiotics are used to infect E. coli. The percent of receptorphage with acquired rVHCH1 genes from the provider vector is expected tobe greater than 5% based on the assumption that each bacteria generates60 phage after overnight culture (Griffiths, Williams et al. 1994). Itis also estimated that as long as this percent of the originaltriantibiotic resistant recovered cells acquires a rVHCH1 chain from theprovider vector, the number of different phage within the rVab librarywill be close to the number of surviving bacteria.

Details of the Individual Steps for Expressing the rVLCL.1.6 andrVHCH1.L.b by CRE-LOX RECOMBINATORIAL FORMATION

Phage P1 lysates are made by thermal induction (Rosner, 1972). E. coliC600 Su- (Appleyard, 1954) containing phage P1Cm c1.100r-m-(Yarmolinsky, Hansen et al. 1989) are grown in a 2 l baffled flaskscontaining 1 l of 2×TY, 25 μg/ml chloramphenicol, 10 mM MgSO₄ withvigorous shaking at 30° C. to an optical density of 0.6 at 600 nm. Thetemperature is then raised quickly to 42° C. by shaking in a 70° C.water bath. Shaking is continued for another 35 min. and then at 37° C.until lysis is visible. Cultures are centrifuged to remove debris andintact cells. Chloroform (100 μl) is added to the supernatant and P1phage after 30 min. 30° C. infection of midlog E. coli TG1 (Gibson,1984) grown in 2×TY broth with 5 mM CaCl₂. Phage infected E. coli aretittered by plating E. coli on TYE medium (Miller, 1972) containing 30μg/ml chloramphenicol. Resistant colonies are counted after 24 hincubation at 30° C. and when expressed as transducing units (t.u.) aregreater than 10⁹ /ml.

One liter of 2×TY broth containing 12.5 μg/ml tetracycline (2×Ty-TET) isinoculated with 10⁹ E. coli carrying the rVLCL.lib cloned in LoxREC(i.e., fdDOG-2lox Vkldel Griffiths, A. D., et.al. 1994). The culture isincubated for 12 h at 30° C. in two 500 ml aliquots in 2 l baffledErlenmeyer flasks. Polyethylene glycol is added to precipitate the phage(McCafferty, Griffiths et al. 1990), which are then suspended in PBS(phosphate buffered saline: 25 mM NaH₂ PO₄, 125 mM NaCl, pH 7.0) andfiltered through a 0.45 μm sterile filter (Minisart, Sartorius). Theresulting phage, are tittered on mid-log E. coli TG1 (30 min, 37° C.)and plated on TYE-TET, (Griffiths, A. D., et.al., 1994) reaches ˜10¹⁰t.u./ml.

The recombination process is monitored by withdrawing aliquots of thephage infected bacteria and serially diluting the bacteria onto TYEplates supplemented with 1% glucose and a variety of the threeantibiotics, ampicillin (100 μg/ml), tetracycline (15 μg/ml) andchloramphenicol (30 μg/ml) and calculating the library size. The rVHCH1library cloned into LoxPRO (i.e., pUC19-21loxVHdel in Griffiths, A. D.,et al. 1994, see above) and contained in about 10⁹ E. Coli, isinoculated in 100 ml 2×TY broth containing 100 μg/ml ampicillin and 1%(w/v) glucose (2×TY:AMP:GLU). An aliquot is taken for c.f.u titering andthe remainder of: the culture is grown overnight at 30° C. A secondaliquot is then taken for c.f.u. titering and one 5 ml aliquot is usedto inoculate 500 ml of 2×TY:AMP:GLU in a 2l Erlenmeyer flask and theculture is grown at 37° C. to an OD of 0.5 (600 nm). To this culture,2×10¹² t.u. of rVLCL.lib in LoxREC is added and the culture is thendivided into 5×100 ml aliquots. Each aliquot is mixed with 1l of2×TY:AMP:GLU, prewarmed to 37° C., and incubated at 37° C. withoutshaking for 30 min, and then with shaking until they reach an OD600 of0.4 (about 30 min). Aliquots are then taken for c.f.u. titering. Twohundred ml of phage P1Cmc1.100r-m- lysate (about 6×10¹¹ t.u.) are addedto each flask (at an m.o.i. of about 1) after the addition of CaCl₂ toobtain a final concentration of 5 mM in CaCl₂. This incubation iscontinued, with short durations of shaking every 15 min. for 1h at 30°C., followed by the centrifugation at 5,000×g for 15 min. The resultantpellets are suspended in 5 l 2×TYB (the original volume) with 100 μg/mlampicillin (100A), 12.5 μg/ml tetracycline (12.5T) and 25 μg/mlchloramphenicol (25C) and 1% glucose (1G). An aliquot is taken forc.f.u. titering and the library size (number of ATC resistant c.f.u.) isconfirmed to be greater than 10¹⁰. An aliquot is centrifuged at 12,000×gfor 5 min. the supernatant filtered through a 0.45 m sterile filter, andthe fd phage titer is determined by infecting log phase E. coli TG1 (30min. 37° C.) and plating on TYE-TET.

The culture, in 5×1 liter aliquots, is incubated overnight at 30° C.(all culturing is with shaking unless specified) for 24 h in 2 l baffledflasks. Aliqouts are taken for bacterial c.f.u. and fd phage (using logphase E. coli TG1) titering with the total yield of fd phage beingconfirmed to be greater than 10¹³ t.u. The culture is centrifuged at5,000×g for 15 min. at 4° C. and the fd phage are precipitated using PEP(McCafferty et al. 1990) and resuspended in a final volume of 10 ml PBS.

Five 2 l flasks, each with 1 l 2×TYB, are inoculated with E. coli TGIand grown at 37° C. until reaching an OD600 of 0.4 (about 4×10¹²bacteria). About 1-2×10¹² t.u. rVab are then added to the 5 l of E. coliand the cultures are incubated without shaking at 37° C. for 30 min. Thenumber of E. coli infected with fd phage is confirmed by platingbacteria on TYE-TET plates to be greater than 10¹². Tetracycline (12.5μg/ml) is then added and the culture is grown for 16h at 30° C. Theculture is then centrifuged at 5,000×g for 10 min. and the pelletcomprising the library is suspended in 250 ml 2×TYB containing 15%glycerol and is stored in 15 ml aliquots at -70° C.

The efficiency of replacement of the endogenous VH to be exchanged inthe phagemid receiver vector LoxREC with rVHCH1 chains from the providervector LoxPRO (i.e., pU19-2loxVHlib) ((Griffiths A. D., et.al., 1994),is determined to be less than about 20% by analyzing 200-300 individualcolonies from the rVablib. Colonies are transferred onto TYE-TET platesand grown overnight at 30° C. Identification of colonies possessing therecombinant VH genes is accomplished using colony hybridization(Tomlinson et al. 1992) with a primer complementary with the CDR3 regionof the exchangeable VH of the LoxREC. Between 40-50 clones lacking theendogenous VH gene (i.e., the antiTNF VH as used in fdDOG-2lox Vdel byGriffiths, A. D. et al., 1994) are screened by PCR (Gussow and Clackson,1989) for the presence of heavy chains with the primers similar toPELBBCK (5'GAA ATA CCT ATT GCC TAC GG) and CH1. LIBSEQFWD (i.e., 5'GGTGCT CTT GGA GGA GGG TGC) and for the presence of light chains with theprimers like fdBCK (5'GCG ATG GTT GTT GTC ATT GTC GGC) and CL.(or CL)LIBSEQFWD (respectively, 5'CAA CTRG CTC ATC AGA TGG CG OR 5'GTG GCC TTGTTG GCTTGA AGC) (Griffiths, A. D., et al. 1994). Both chains areexpected to appear among the clones at frequency of about 20-30%.

Aliquots are then spread on TYE-TET in dishes (Nunc), and are incubatedovernight at 30° C. as well as being tittered by serial dilution onsmall TYE-TET plates to allow determination of the number of colonies onthe large plates. The plates containing the necessary bacteria togenerate 10⁷ clones are accumulated, and the bacteria are scraped into10 ml 2×TYB containing 15% glycerol to make stocks corresponding to rVablibraries of greater than10⁷ clones.

XII. Step 5--Generating Phage and Displaying the rVab.lib on PhageSurfaces (FIG. 14)

As constructed above, each phagemid carries and expresses an individualmember of the rVab.lib. As shown in FIG. 14, VHCH1 protein is expressedas a fusion protein coupled in open reading frame to the NH2-terminus ofthe fd gpIII coat protein gene and is therefore displayed on the maturephage surface as an attached surface protein. The VLCL protein,expressed via appropriate leader and double terminator codons as asoluble protein, is released into the bacterial periplasmic spacewherein under reducing conditions it spontaneously forms activedisulfide linked dimmers with VHCH to produce the desired functionalrecombinant rVab on the surface of the mature phage. Phage lysatesexpressing the entire combinatorial rVab library (one rVHCH and onerVLCL gene per phage) are made with the aid of helper phage.

Phage, helper phage, plasmid construction, and titering are as generallydescribed in the literature and phage and helper phage are availablefrom commercial sources (Stratacyte CA, or Cambridge AntibodiesTechnologies, UK). The lysates are in general made as follows: five l of2×TY-TET is inoculated with a 15 (5-20) ml aliquot of the rVab phagelibrary (greater than 2×10¹⁰ c.f.u.), the cultures are grown overnightat 30° C. in baffled flasks (1 l medium/fl), centrifuged at 5,000×g for15 min at 4° C. and the fd phage are precipitated with PEP (McCaffertyet al. 1990). Phage is then resuspended in a final volume of 10 ml PBS.These lysates are designated rVab.lib.F and have total yields of rVabexpressing nature phage of from 10¹³ to 10¹⁴ t.u.

EXAMPLE 2 Preparation of SOMERs For The Human Type 1 MuscarinicAcetylcholize Receptor

In this example, following Stages I and II of the TSA process (FIG. 1 ),rVabs from the rVab.lib are identified, isolated and used to establishan assay for small organic molecules (SOMER) which bind to and regulatethe activity of only one subtype of human muscarinic cholinergicreceptor (huAChRm). Such SOMERS are useful new discovery leads for suchdiseases as Alzheimer's and other memory and learning deficits. Thesteps outlined below constitute Stages I-II (see FIG. 1) of the processof the invention and are those necessary to isolate from the rVab.libthose rVab members which bind (T+) to type 1 of the AChRm subtypes,regulate its activity (A+), and are specific and selective (S+) forsubtype 1 of the human muscarinic receptor (huAChRm1). Stage III of theinvention, using these TSA+ rVabs to generate 3D models ofACHRm1-specific pharmacophores (BEEPS, see below) and obtain SOMERs isbriefly outlined at the end.

Stages I-II detail the steps necessary to obtain and use the specific.AChRm1 rVab to establish simple rapid radioreceptor assays for smallorganic molecules (SOMERs) which specifically bind and regulatehuAChRm1. As disclosed herein, and illustrated in FIGS. 18 and 19, theserVabs are used to discover active surfaces on the huAChRm1which are notpresent on the other huAChRm2-5 subtypes. In addition, the rVabs may beagonists or antagonists at selective huAChRm subtypes (i.e., m₁₋₅) andmay exhibit specificity(S+) of action between one m subtype and theother four.

Phase I of this process reconstitutes functional huAChRm which are thetarget of these assays. Phase II first identifies the rVabs containedwithin the rVab.lib which bind to huAChRm1 (i.e., are T+), and areselective among the five huAChRm subtypes (Andre, Marullo et al. 1987)as well as specific for huAChRm over non-cholinergic neurotransmitterreceptors. In this example these two attributes are referred together asS+. Subsequently, Phase II identifies and isolates the subpopulation ofTS+ huAChRm rVab which regulate the activity of the huAChRm1(A+) withsimilar TS+ attributes. The rVabs with all these attributes are referredto as TSA+ rVabs. Phase III converts the TSA+ rVabs to reporters (i.e.,rVab.reporters) and establishes validated automated rapid receptorbinding screens for small organic molecules (SOMERS) which competitivelydisplace active rVab reporters from active surfaces on huAChRm1. Amongthese SOMERS are those having the desired activity profile of apharmaceutical discovery lead, i.e., selective specific regulation ofAChRm1.

Phase I-A: Obtaining AChRm

Cortical membranes enriched in huAChmR are prepared from brains (freshor frozen, human, porcine or bovine) as outlined by Haga & Haga (Hagaand Haga 1983). Membranes are prepared by homogenization in standardfashion (i.e., with protease inhibitors) and AChRm is solubilized bytreatment with 1% digitonin, 0.1% NaCholate in 50 mM NaCl/buffer. Thesoluble receptor is purified over an 3-(2'-amino benzhydryloxy) tropane(ABT) affinity column and is eluted from the ABT column by atropine.Soluble receptor is subsequently applied onto a hydroxyapatite column toremove the free atropine. The receptor is then eluted with highpotassium phosphate and 0.1% digitonin and is further purified through asecond round of ABT purification as rioted above. Two rounds of HPLCpurification over tandem linked TSK4000SW and TSK3000SW columns providesthe final purification and the receptor is suspended in 0.1 M potassiumphosphate with 0.1% digitonin.

As a secondary source, the five huAChRm1-5, expressed as recombinantproteins (rhuAChRm1-5) in Sf9 cells containing an expression vectorbaculovirus construct carrying one of the huAChRm as originallydescribed by Vasudeva (Vasudevan, Reilander et al. 1991) are obtainedfrom commercial sources (BioSignal, Inc., Montreal, Canada). Otheralternative sources of huAChRm are various tissue culture cell linestransfected and expressing cloned huAChRm (Kubo, Fukuda et al. 1986;Shapiro, Scherer et al. 1988; Buckley, Bonner et al. 1989; Buckley,Hulme et al. 1990; Tietje, Goldman et al. 1990; van Koppen and Nathanson1990; Kashihara, Varga et al. 1992; Beth 1993; Lazareno, Farries et al.1993; van Koppen, and Lenz et al. 1993).

Phase I-B: Obtaining the G proteins (GP)

Go, Gi and Gn (referred to as G protein [GP] in text and G in Figures)are purified as described (Sternweis, 1984; Haga, 1986, and Haga,Uchiyama, et.al., 1989). Brains (150 g), porcine, bovine or human(obtained from commercial or non-profit sources) are homogenized, themembranes pelleted and then solubilized with 1% NaCholate in 20 mMTrisHCl (pH 8.0) 1 mM EDTA, 1 mM DTT (1%Cho-TED) with 0.1 mM benzamidine(2 L vol.). After centrifugation, the supernatant is applied to DAESephacel and the fractions binding [³⁵ S]GTPS are eluted with linearNaCl, in 1%Cho-TED, concentrated, and applied and eluted from UltrogelAcA 34 in 0.1M NaCl in Cho-TED. The fractions with [³⁵ S]GTPS bindingactivity are pooled with TED+0.1M NaCl (450 ml) and applied toheptylamine-Sepharose, washed and finally are eluted with a lineargradient of 0.25% NaCho-TED+0.2M NaCl vs. 1.3% NaCho-TED+0.05M NaCl.This material (a mixture of Gi and Go) is applied to DEAE-Toyopearl,prewashed with TED+0.6% Lubrol PX (0.6%LPX-TED) and eluted with a lineargradient of NaCl in 0.6%LPX-TED. The Gi fractions elute first, then theGo fractions. Each is collected separately and is stored at -80° C.until use. Before use, the Lubrol is changed to 0.8% NaCholate, inTED+0.5M K phosphate buffer pH7,0.1MNaCl) on a small column ofhydroxyapatite.

Phase I-C: Reconstitution of an active AChRm:GP complex

Reconstitution is accomplished as per Florio and Sternweis (Florio,1985). Porcine [or human brain total lipids: as per Folch, J., Lees, M.,and Stanley, G. H. S. (Folch, Lees et al. 1957). The lipid mixture isprepared (Haga, 1986) from brain extract (Folch fraction I) (1.5 mgeach) and total lipids (1.5 mg each) suspended in 1 ml HEN (20 mMHepes-KOH buffer pH 8.0, 1 mM EDTA and 160 mM NaCl) with 0.18%deoxycholate and 0.04% sodium cholate. rhuAChRm (0.2-0.4 nmol/ml [³H]QNB binding sites in PD (0.5M potassium phosphate buffer pH 7.0 and0.1% digitonin (10-40 μl)) are mixed with 0.1 mM oxotremorine in HEN,and then with 100 μl of lipid mixture (final vol. 200 μl) to give QNB:Rcomplex. The complex is then run through a Sephadex G50 column and thevoid volume (1-8 pmol [³ H]QNB binding sites, 400 μl) is collected. ThehuAChRm:QNB complex is mixed with G protein (mixtures or separateG-proteins, 0-200 pmol of [³⁵ S]GTPgS binding sites in 40 μl cholatesolution) CN-TED and HEN (50 μl) containing MgCl₂ and DTT (finalconcentration 10 and 5 mM respectively) and incubated at 0° C. for 1 hr.This huAChRm1:GP mixture is diluted before use with p3-5 vol of HEN.

Phase I-D: Attachment of active huAChRm to matrices (FIG. 19)

huAChRm (abbreviated AR in text and R or T in Figures), alone orcomplexed with GP, is attached to a Sepharose (or agarose)-type matrixby taking 5 ml of matrix (WGA-Sepharose, mmolWGA/ml Sepharose, 50% v/v,prewashed and suspended in buffer A (25 mM Potassium phosphate buffer,[pH 7.0 ], 0.8 mM EDTA, 10 mM MgCl2, 230 mM NaCl, 0.06% BSA, and 4 mMHEPES KOH buffer (pH 8.0]) and mixing it with less than 1 mlreconstituted AR:GP complexes (100 pmol AR/ml). The mixture is thenincubated at room temp (r.t.) for 30 min, diluted with buffer A to 20 mland the Sepharose is allowed to settle (or centrifuge at low speed[5,000×g, 1-2 min]). The Sepharose is then resuspended in 20 ml buffer Aand the washes are repeated twice to provide purified ARcomplexed-Sepharose WGA [sWGA:ARGP] material. Recombinantly derived ornative AR:GP complexes with appropriate sugar residues bound to WGA inthis process remain active as matrix-attached receptor in agreement withpublished data showing glycosylation is not required for AChRm activity(Habecker, Tietje et al. 1993). Quantitation of bound AR:GP to sWGA isverified by [³ H]QNB ±10 μM atropine and [³⁵ S]GPTS or [³ H]GppNHp ±0.1mM GTPS or GppNHp binding using standard binding assays (Berrie,Birdsall et al. 1985; Haga, Haga et al. 1986; Wheatley, Hulme et al.1986; Poyner, Birdsall et al. 1989).

In parallel reactions, AChRm (or GP), natural or recombinantly expressedpreparations, are attached by standard techniques to plastic, directlyor secondarily, through matrix attached antibodies, naturally derived orrVab-type, which recognize epitopes on the receptor, glycoprotein,G-protein or small peptide tags (i.e., the c-myc and other amino orcarboxy terminal in frame tagging peptides, available in various spacedcommercial expression vectors). After attachment of AR, the unoccupiedreactive matrix surfaces are blocked by application of various standardblocking agents (i.e., BSA, milk etc.).

Phase II: Panning for TSA+rVab

In this stage, rVabs which possesses TSA+ attributes are identified a sthose which bind to AChR directly or indirectly attached to the matrix,with or without G, in buffer conditions similar to those used for AChRmradioreceptor binding studies. These conditions maintain receptoractivity. In all cases plastic and not glass is used for directattachment matrix surfaces and reaction vesicles to minimize rVabnonspecific absorption to glass. The buffer for these reactions is a 10mM potassium phosphate (pH 7.0), 0.8 mM EDTA, 10 mM MgCl₂, 0.230 mMNaCl, 0.06% BSA, 4 mM Hepes-KOH (pH 8.0) buffer, and optionally furthercomprising guanine nucleotide (GTP) and/or muscarinic agonist orantagonist as detailed below. This stage isolates four types of A+ TSA+rVab antibodies: agonist like (Ago+), partial agonist-like (partAgo+),allosterically agonist (Alloago+) and antagonist-like (Antago+)(outlined in FIG. 19).

Phase II-A: Panning for receptor [Target (T⁺)] recognition

The general process is summarized in FIG. 16 and the specificapplication in FIG. 19: Five ml of the rVab.lib (10¹¹⁻¹² PFU/5 ml, andsuspended in buffer) is mixed with 1.0 ml settled s-WGA:GAR in buffer A,and incubated at 30° C. with gentle shaking for 60 min. The mixture isthen centrifuged at low speed (LSS) of 500×g for 15 min. The supernatantis decanted and diluted with buffer A to 10 ml. These washes arerepeated 3 times rapidly and the rVab in the final pellet resuspended inbuffer A and designated as the T⁺ rVab.lib. (FIG. 19). Phage arereleased by elution with 100 mM triethylamine (Marks, Hoogenboom et al.1991). Aliquots are withdrawn and tittered for phage. The population ofisolated phage are then amplified by infection and induction of newlysates and panned again 2-4 more times to generate the final T+rVabpopulation of phage for subsequent isolation of the four types ofA+rVabs.

Phase II-B; Panning for Active rVabs (A+rVab) (Phase IIB)

In this process (general outline in FIG. 17, specific application inFIG. 19), the subset of rVab from the amplified T+rVab population whichare potentially agonistic are induced by the addition of guaninenucleotides to dissociate from the matrix attached R:G complex and beisolated as free TA+rVab in the supernatant. In this process, the rVabwhich bind and act as antagonists, or bind to nonactive surfaces, remainmatrix-receptor associated after the addition of guanine nucleotide. Thenegative influence of GTP on T+rVab binding is taken as indicative ofpotential agonist action of the bound rVab based on the observation thatin functionally coupled AR:GP complexes there is a negative reciprocalinteraction between the binding of GTP or GDP to the G protein, andagonist to the receptor, which can be observed as an immediatedissociation of either from the complex (Smith, Perry et al. 1987;Poyner, Birdsall et al. 1989; Lazareno, Farries et al. 1993). No suchreciprocal interactions occur between antagonist and guanine nucleoticlebinding (Buckley, Bonner et al. 1989).

The TA+ rVab released into the supernatant are further separated andisolated as one of three types of agonists in separate panning steps(see below Phase IIB-i,ii,iii). The specific muscarinic activity of therVab is confirmed at the end of all isolations using AChRml activityassays in which potential TSA+rVabs (a) compete with radiolabelledantagonist (or agonist), (b) dissociate prebound [³⁵ S]GPTS or [³H]GppNHp from matrix bound AChRm:GP complexes, (c) stimulate GTPase andor GTP exchange, and d) regulate the activity of other effector systemscoupled to the AChRm1 (i.e., adenylate cyclase, phospholipase, Kchannels) in various published in vitro, cellular or animal assaysystems (Yatani, Mattera et al. 1988; Fraser, Wang et al. 1989; Shapiroand Nathanson 1989; Kobayashi, Shibasaki et al. 1990; van Koppen andNathanson 1990; Weiss, Bonner et al. 1990; Yatani, Okabe et al. 1990).

In Phase IIB, addition to bound T+rVab of ACh itself can also be used,via the same type of induction of rVab dissociation from AChR, toisolate those rVab which bind not to the ACh binding pocket but to GP atactive nucleotide binding surfaces or to other surfaces on AR or GPwhich are active and allosterically connected with the cholinergicbinding surfaces of the AChRm1.

Specifically, at the start of Phase IIB, the amplified T+rVab.libisolated in Phase IIA is mixed with matrix-bound AChRm1:G complex, in 10volumes buffer A as noted above for 30 min at 37° C. The pellet iscentrifuged at low speed, resuspend in 10 vol cold buffer A andimmediately recentrifuged. The washed pellet is resuspended in 10 volcold buffer A containing 100 uM GTP. After less than or equal to about 1min. the matrix:AR:GP complex is centrifuged at low speed, and thesupernatant is separated from the pellet to be used to isolate threedifferent types of agonistic rVab in Phase IIB-i,ii-iii. The pellets arewashed in similar fashion with buffer A three (3) times and analyzed inphase IIB-iv for muscarinic antagonist (Antago+) activity as detailedbelow. Throughout these phases, aliquots of supernatant are taken totiter the phage, and if less than 10⁶ /ml, the phage are amplified andrecycled as above 2-3 additional times. To the final supernatant,containing rVab induced to dissociated via GTP addition, GTPase andGDPase are added and the supernatant incubated 30 min at 30° C. Thesolution is then chilled and passed over a Sephadex G50 fine columnusing buffer A and the void volume, free of any remaining nucleotides,is taken and labeled TA⁺(GTP+) rVab.lib.

Identification of Antagonist Activity

The T+rVab lib, for which binding is not modified by addition of GTP,and which is recovered bound to matrix in the presence of GTP, isreleased from matrix and the phage harvested by PEE, precipitation inPhase IIB-iv. The phage are then resuspended and mixed with s-WGA:RG in2 ml buffer A containing saturating amounts of antagonist (atropine 10μM, perenzepine 1 μM, scopolamine, 1 μM). Following incubation for 60'at 30° C. phage and s-WGA:RG are centrifuged at low speed and thesupernatant is collected. The free phage are isolated, and amplified (asnoted above) and the population recycled an additional 2 to 3 times bycombining with s-WGA:RG to remove from the supernatant phage which, inthe presence of antagonist, do not bind to s-WGA:AR. The phage in thefinal supernatant contain the expressed A+rVab members which aremuscarinic antagonist-like (Antago+) are designated at the end of PhaseIIB-iv, as TAntago+ huAChRm1 rVab.lib. [see FIG. 19, rVab-4].

The pellet from incubation with muscarinic antagonist in the above PhaseIIB-iv contains a T+rVab sublibrary which has members which interactdirectly with surfaces on the G protein of the AR:GP complex and areguanine nucleotide like regulators of the AChRm1:G complex. Phage arefreed from the matrix, amplified and incubated with matrix boundG-protein in buffer A. The matrix, and attached rVab, are thencentrifuged, washed and attached phage isolated. Confirmation of G-likeactivity among these isolated rVabs is done in standard radioreceptorbinding assays establishing competition with radiolabelled GppNHp orGTP.sub.γ S for binding to GP.

Phase IIB-i,ii,iii: Separating GTP Sensitive A+rVab into Ago+(^(GTP+CCh)sensitive) and alloAgo+(^(GTP+) CCh-insensitive) AChRm1-rVab (FIG. 19 )

One to 10 ml of the TA⁺(GTP+) rVab.lib is mixed with 1 ml sWGA-GR,incubated 60' min, 30° C. in buffer A with 300 μM stable muscarinicagonist carbachol (CCh) and is then centrifuged at low speed. In PhaseIIB-iii, the pellet is washed with buffer A three (3) times, andresuspended in buffer A and the phage isolated in standard fashion. Thisphage population, labelled TA⁺(GTP+CCH-) rVab.lib, contains theallosterically acting muscarinic like agonist (alloAgo+) rVab members(FIG. 19, rVab-3).

The supernatant from the above Phase IIB incubation with CCh is passedover Sephadex G50 (fine) in Phase IIB-i,ii and the phage are collectedin the void volume of the column (as outlined above) to obtain CCh freerVab which are blocked from binding to AChRm by CCh. These phage arelabeled as the TA⁺(GTP+CCH+) rVab.lib and contain the Phase IIB-i and iirVab.lib members which are competitive-ACh muscarinic full(i) orpartial(ii) agonist-like (Ago+) antibodies (i.e., rVab-1 and 2 in FIG.19).

Phase II-C: Separating Selective(S+) from non Selective(S-) TA⁺ rVabs

All four types of AChRm A+T+rVab phage isolated in Phase IIB (label-LedrVab-1,2,3 &4 FIG. 19), are taken separately, and mixed with 1 mlsWGA:GR m1 in buffer A containing soluble complexes of GP and AChR ofsubtypes 2-5 (i.e., G:AChRm2-5 complexes). These complexes are added asthe competing target peptide (analogous to comp-T-pep in FIG. 16) whichcontain greater than 10 fold excess of surface epitopes which are not tobe recognized by the ml specific A+rVabs, incubated 30° C., 60 min andthen centrifuged at low speed(FIG. 19). The pellets contain theS+rVabs.lib members and these are resuspended in 10 vol buffer A andwashed immediately. The phage are recovered in standard fashion,amplified and cycled through Phase IIC two to four additional times.Frozen stock bacterial cultures and phage lysates are prepared for eachof the four A+ types of AChRm1 specific(S+) and are designated TS(Ago;partAgo; alloAgo; or antAgo)+ rVab.lib. In an alternative embodiment,isolation of the AChRm1 specific rVab library is done on the T+rVab.libbefore selecting for the A+rVab.lib (FIG. 16) and the population isamplified for subsequent A+ selection as defined above.

Stage II-E: Confirmation of A+ activity among individual members of theTSA+ rVab AChm1 lib

Individual members (10-20) of each of the four A+ type TSA+ rVab AChRm1library identified above are obtained and phage lysates are generatedfor each by standard technology. The A+ profile for individual phagemembers of each of the above four A+ library is confirmed andquantitated by a nM ED50 value in one or more of the following standardradioreceptor and receptor-coupled activity assays. The radioreceptorassays use 1) active soluble targets (i.e., AChRm, AchRm:G and G-proteincomplexes); 2) radiolabelled AChRm [³ H]agonist or antagonist, or [³ H,or ³² P]GTP, or GMPPNP or [³⁵ S]GTPS in buffers used for rVab isolation;and 3) various dilutions of individual rVab members to be tested. Thereaction mixture contents are incubated at 30° C. for 30 min and thetargets are recovered free of soluble radioligand by standard filtrationor PEG precipitation. The reduction in specifically bound radiolabel isthen quantitated.

The degree of agonist activity for Ago+, partAgo+ and alloAgo+ rVabmembers is demonstrated by dose response alteration of any one of anumber of AChRm1 coupled effector systems. Individual antagonism(Antago+) is demonstrated by dose response blockage of the ACh agonisteffect on the particular receptor coupled system.

Phase III. Conversion of Selected A+ rVab to rVab Reporters

A. Preparation of Reporters and Competitive Binding Assays to IdentifySOMERs (FIGS. 18,19)

DNA is isolated from phage lysates prepared from bacteria grown from twoto five individual TSA+rVab.bact stocks from each of the four classes ofA+ libraries characterized above to have A+ activities with ED50 valuesof 1-30 nM. The DNA is digested with ApaL1 and Not1 to release from thefd.o slashed.Carrier the rVLCL-rVHCH1 rVab construct. One μg of theinsert is isolated and mixed with 5 μg DNA from pEXPRESSORrVab(pEXPRESSORrVab-1, see FIG. 9), precut with ApaL1 and Not1, and 1200U T4ligase (Sambrook, Fritsch et al. 1990). The ligated products arepurified and electroporated into E. coli (Dower, Miller et al. 1988).Transformants are grown and characterized by diagnostic PCR and thensequenced. Correct constructs of each are then grown, the recombinantrVab (i.e., VHCH1: VLCL dimmer chains) induced and the rVab products arerecovered in the supernatant by precipitation with Sepharose coupled VHor VL chain antibodies or antibodies to peptide sequences (ISOTAGS)included in pEXPRESSORrVab-I (FIG. 9C) and fused in frame to thecarboxyterminus of CH1. The rVab are then released from theprecipitating antibody. The VHCH1 chain of the rVab is thenphosphorylated in a constant region C terminal domain attached in frame(Li, et al. 1989) when rVab is ligated to pEXPRESSrVab. Thephosphorylation reaction uses protein kinase and [³² P]ATP followingpublished methodology and the radiolabelled product is isolated in thevoid volume of a G50 column. The radiolabelled rVab is mixed with BSAand stored at -4° C. until use.

To establish a saturation isotherm and ED50 for the labelled rVab withits active target (soluble or membrane bound; GP, AChrR m1, orAChrRm1:GP complexes), the binding of rVab is determined from reactionmixtures (50 μl) comprising from 1000-1,000,000 cpm of radiolabelledrVab with and without 1000 folded excess of unlabelled rVab in buffer B.Identical control assays are done with AChRm2-5, AChRnicotinic, or othernon-cholinergic G-protein linked neurotransmitter receptors (e.g.,beta-and alpha adrenergic, and opiate receptor). These assays areincubated for 30 min at 30° C. The [³² P]rVab:target complex is PEGprecipitated (or filtered with membrane bound target) and counted forradioactivity.

The induced dissociation of rVab from its target by an allostericeffector (i.e., the Ago+rVabs with GTP) defines the class of allostericrVab agonists. A series of competition binding assays is then performedusing less than, or equal to, the ED50 amount of [³² P]rVab withincreasing concentrations of the nonlabelled form of the same rVab,other rVab, standard muscarinic specific ligands (agonists andantagonists), and a number of noncholinergic ligands as controls tofurther characterize these rVabs.

These assays establish a saturation binding isotherm, an apparent Kd forrVab and target association, and IC50 values for various ligands andother rVabs. The reactions carried out in the presence of increasingconcentrations of other members of the same TSA+ rVab group define therVab with the lowest IC50 value. This rVab is then converted to aradiolabelled form for use in obtaining saturation isotherms and variouscompetition curves. In addition to the radiolabelled rVab, these assaysfurther may contain 1) target agonist; 2) antagonist; 3) GTP; and 4)combinations of all three. Standards such as nicotine, muscarine, ATP,GMP, and the various small organic molecules previously reported in theliterature to have affinity for regulation of AChRm receptor of the m1-5type regardless of affinity or selectivity may also be included.Saturation isotherms are generally conducted over a concentration rangeof four to six orders of magnitude.

rVab's with affinity for AChRm1 of less than about 10 nM, selectivityfor AchRm1 over AchR types m2-5of >100 fold, and specificity regardingnon-cholinergic soluble receptors of 1000 fold are appropriate asrVab-REPORTERs for A+ activity for use in Stages II and III of thisinvention wherein SOMERs are identified in CHEMFILES or synthesizedbased on BEEP models (see below).

Phases IV-VI

In the last three phases of the invention, which are part of TSA StageIII, the TSA+ rVabs are grouped according to common epitopes andattributes (Phase IV), 3D-models of active pharmacophores (BEEPS) arederived (Phase V) and the pharmacophores used to find SOMERs in existingCHEMFILES or by synthesis (Phase VI). The grouping of TSA+rhuAChRm1 inPhase IV is accomplished according to a) the common surfaces recognizedby the rVab (defined by competition by peptide fragments of the AChR; b)the type of activity exhibited by the rVab (partial or full agonist,antagonist, competitive or allosteric with ACh or GTP) and; c) thediversified amino acids of the V regions found in the rVab.

The Stage III analysis of the TSA+rVabs which creates a 3D modelpharmacophore (FIGS. 23-25) is performed based on a genetic algorithmdirected comparison of the array and positions of the amino acids in theV regions of the active rVab's, including CSR, CDR and frameworkresidues. The 3D atomic model formulated by this process is designated a"biologically enhanced ensembled pharmacophore" (BEEP). The BEEPcontains sufficient information to describe the elements of a SOMERnecessary for the activity profile of the active rVabs within thatparticular group.

In Phase VI, the BEEP is used in a variety of available programs (HOOK,LOOK, and DOCK) for computational screening (Phase VIa) of availableCHEMFILES for huAChRm1 SOMERs and, in a rational drug design effort, todirect the actual synthesize of huAChRm1 SOMERs (Phase VIb). SOMERsobtained by either approach are then confirmed as TSA+ AChRm1 agonistsor antagonists in in vitro, cellular and animal assays, known to thoseversed in cholinomimetics.

Additional diversification of TSA+ rVabs within CSRs and CDRH3 iscarried out by PCR (as detailed in the construction of the originalrVab.lib) in Phase IVb whenever the number of rVab within a group isless than 10 or when sufficient information is not obtainable from thenumber of A+ rVab's identified to develop BEEPS with the desiredusefulness for identifying SOMERs and simplification of the TSA+population is done when the number of rVab within a group is >100 (FIG.15)

EXAMPLE 3

This example outlines the TSA process establishing simple competitivebinding assays for multimeric small organic molecules, which in thisexample are DISOMERs, capable of regulating the activity of growthhormone receptor. Here, DISOMER discovery is based on the discovery ofpairs of rVab which identify active surfaces on Growth Hormone Receptorand their conversion to rVab.REPORTERs according to the method of theinvention.

This methodology establishes a generic approach for discovery of drugsactive at oligomeric receptor targets, or targets requiring activationat multiple sites of a monomeric unit. In such systems the "receptor" isdefined by multiple surfaces which must be in contact with the signal tocause activation.

The process of this invention provides a means of identifying activeligands for multiple site receptors a) which have more than one activesurface; b) more than one subunit per active receptor complex; or c)different subunits and active surfaces. This method is also suitablewhere more than one subunit contains a portion of an active surface; thesurface required for activation is too large to be occupied by a singlesmall organic molecule present: within a CHEMFILE; and activation ofoligomeric receptors is intimately associated with the hormone inducedformation of complexes of at least two receptor subunits (Cunningham,1991; Kelly, 1991; DeVos, 1992; and Wells, 1993).

Unlike standard screens to identify a single chemical entity to replacea large multi-site binding hormone, the approach described according tothis invention, identifies pairs of active surfaces, finds SOMERs foreach individual active surface, and then links the SOMERS together tocreate multimeric units (e.g.,DISOMER) large enough to replace themultivalent hormone, e.g., growth hormone (GH). In the example provided,the target oligomeric receptor is the homo-dimeric growth hormonereceptor (GHR) and the active surfaces identified are the two surfacesused by GH for active GHR dimerization. For GHR there is only one typeof receptor subunit, referred to here as T1. Activation of the receptorrequires GH to dimerize two receptor subunits (T1²) by maintainingbinding of active surfaces on two T1.

1. Identification and Isolation of rVabs Specific for GHR

Step 1a: Identification of GHRT+rVab.lib for the T1 GHR Subunits

Isolate from the rVab.lib the subpopulation which binds to the surfacesof the T1 GHR subunit. These rVabs are designated GHR.T+rVab.lib.

Library surface scanners are provided by the rVab.lib constructed asoutlined in Example 1 of this invention. This rVab.lib, i.e., rVHCH:VLCLcomplexes, is expressed on phage surfaces attached to the phage gpIIIcoat protein. A one ml aliquot of phage lysate (>10¹² t.u.) is mixedwith GHR receptor subunits (T1) which are prebound to an immolized solidsupport i.e., agarose bead-type isolation matrix (mat-T1). In thisexample, the basic GHR subunit (T1) used is that which encompasses onlythe excellular domain of the hGHR, including hGHR amino acids 1 to 238(Leung, 1987; Fuh, 1990) with an unpaired penultimate cysteine (Bass,Greene et al. 1990). This form is referred to as sGHR and is expressedin E. coli as an extracellularly released soluble protein (Fuh,Mulkerrin et al. 1990). This soluble protein is then purified (Fuh,Mulkerrin et al. 1990) and bound to beads or plastic through itsunpaired cysteine (Bass, Greene et al. 1990), or to plastic through anantibody which recognizes the sGHR but does not interfere with GHbinding or active GHR dimerization (Fuh, Mulkerrin et al. 1990;Cunningham, Ultsch et al. 1991). All forms of sGHR bind GH as does theendogenous membrane associated entact GHR (Leung, 1987; Fuh, 1990). Anexcess of soluble prolactin receptor (PRLR) as competing peptide(comp-T-peptide) (see FIG. 16) or various mutant hGHR, or PRLR missingeither H binding site I or II (Cunningham, 1991; DeVos, 1992; andRozakis-Adcock, 1992) to compete binding of non-specific rVab binderswhich have no selectivity for GHR binding is routinely added to themixture to define rVab specificity. With sGHR attached to 0.2 mg ofoxivane polyacrylamide beads (Sigma) the reaction mixtures can be assmall as 50 ul beads. The excess of soluble prolactin receptor competesfor binding of non-specific rVab binders which have no selectivity forGHR binding. The mixture is incubated for at least 3 hr at 30° C. inbuffer A which supports normal GHg and GHR association with one entitydisplayed as an attached phage coat protein (Bass, Greene et al. 1990)and consists of <50 mM Tris, pH 7.4, 1 mM EDTA50 mM NaCl, 1 mg/ml BSAand 0.02% Tween 20 and washed three (3) times in 30C buffer A. The rVabbound to the matrix associated GHR, in the presence of the excesscompeting soluble non-GHR related peptide (i.e., the comp-T-pep) isdesignated the GHRTS+ rVab.lib. The phage are recovered by washing (2×)either in Buffer A with 20 nM hGH or 0.2M glycine (pH2.1) (Bass, Greeneet al. 1990) and tittered.

The phage libraries are mixed with E. coli (at a multiplicity ofinfection) of approximately one (1), incubated without shaking for 30min and then plated in antibiotic media and grown overnight andtittered. The survivors are pooled and grown overnight and frozen asbacterial stocks, in 15% glycerol. An aliquot of the stock is grown upand new phage lysates are made and tittered. This phage population,GHR.TS+rVab recognizes all surfaces on the T1 subunit of GHR. Definitionof S+ in this population at this time is not mandatory, and can beomitted, i.e., by not adding prolactin receptor (or any othercomp-T-pep) to the original reaction mixture above, if the number ofGHR.TS+rVab members obtained in Step 1 which are competed by GH (seebelow) is less than 100.

An additional phase of V region amino acid diversification within CSRsand/or CDRH3, as per outlined in the Example 1 and summarized in FIG.15, is performed if greater numbers of GHR.T+ or TS+rVab are desired.

Step 1b: Subdivision of TS+rVab based on GHR Surface Epitope Recognized

1b) Group library members according to common receptor surfacesrecognized. Designate groups as GHR(x-y).T+rVab.lib, where x-y is theamino acid domain of the T1 unit containing the common group epitope(FIG. 16).

Separation according to the receptor surface recognized is accomplishedby adding aliquots of TS+rVab to plastic dishes to which have beenpreabsorbed peptides (obtained commercially) of 10-20 amino acidoverlapping amino acid sequences of GHR and those domains containingamino acid sequences known to influence GH binding (i.e., hGHR aminoacids 54-68, 171-185, 9 [GHR siteI]); and 116-119 and 8-14 (GHRsiteII)as described (Cunningham, Henner et al. 1990). TS+rVab are incubatedwith preadsorbed peptides in buffer A (20 mM TrisHCl buffer pH 7.5, 1 mMEDTA, 0.1% bovine serum albumen) for 3 hr at 30° C. The dishes arewashed to remove unbound rVabs. Bound rVabs are released from thematrix, tittered and amplified again via infection in E. coli. Bindingto these overlapping GHR peptides produces a grouping according toprimary receptor amino acid sequence and hormone binding. Each of theseparate groups are then mixed with soluble matrix-GHR (see step 1a) inbuffer A with greater than 100 fold excess GH and incubated 3 hr at 30°C. and centrifuged. The phage in the supernatant are tittered, amplifiedand further enriched by panning 2-3 additional times for TS+rVabs whichdo not bind to GHR in the presence of GH. This recycling produces apopulation of GHR.TS+rVab which bind to a surface of the GHR which isnormally occupied by bound GH. Although these steps do not identifyand/or subdivide all GHR hormone related epitopes, they divide theoriginal GHRTS+rVab.lib into workable sized subgroups based on bindingto various amino acid sequences and domains involved in GHR recognition.Each group is tittered, amplified, infected into E. coli and bacterialstocks and subsequent new phage lysates are prepared. Each group isdesignated by its amino acid receptor sequence or domain recognized(e.g., amino acid x-y) as follows: GHR.T(x-y)S+rVab.lib. Competition bythese rVab for I125hGH binding to sGHR is done in standard bindingassays (Spencer, Hammonds et al. 1988) in buffer A with terminated byprecipitation by polyethylene glycol 8000, at 4° C. in phosphatebuffered saline as described (Leung, 1987). Competition binding tomembrane associated GHR is performed under identical conditions andreactions are terminated by filtration and washing.

2. Formation and Identification of Bifunctional Active rVabs PossessingRandom Sequences of Amino Acid

Step 2a: Preparation and Expression of rVab-Pep Library

2a) Attach a random 8 amino acid peptide library (Pep 8) in frame to thelight chain (VLCL) of all members of a rVab library recognizing a commonGHR surface (FIG. 11 and 20). Designate these bifunctional surfacebinder libraries GHR(xy).T+rVab-pep.lib.

Each of the group libraries is genetically engineered to be expressed,in a coupled manner, with a short random peptide of 8 amino acids (pep8) attached through a short linker (LNKR) to one chain of the rVab (FIG.11). Attachment can be at different positions on different chainsdepending upon which Cre-Lox recombination system is used to combine therVHCH1.lib and rVLCL.lib onto the same piece of DNA when the rVab.lib ismade (see FIG. 11 vs. 13). In this example, the rVab.lib is madeaccording to Example 1 (FIG. 11) and attachment of the pep 8 is to theamino terminus of the VL region of the rVLCL.lib (FIGS. 11 and 20). InExample 4 below, the construction of a different rVab.lib where additionto a single pep8 could be made to either the carboxyterminus of theconstant domain (CL) of the rVLCL or to the aminoterminus domain of theVH of the rVHCH1 is described (also see FIG. 13).

In this example, attachment is accomplished by using PCR to append thepep8 library to the 5' end of the VL region within the rVLCL members ofthe GHR.TS+rVab.lib. This reaction uses forward primer CH209-216-Not1FWDand back primer APAEPEP8LNKRBCK (i.e., leader seq.Apa1-(NNN)₈ (GGGGS)₁VL1-7) (see Primer Table, FIG. 10). These reactions contain an aliquotof bacteria from each GHRT(x-y)S+rVab.lib., Taq polymerase and forwardand back primers and are cycled 25 times (94° C. 1 min, 60° C. for 1 minand 72° C. for 2 min). The amplified, appended DNA is purified usingMagic PCR PREPS (Promega) and after suspension in water, 1 μg of thepurified DNA is digested with Not1 and Apa1 and ligated using 1200U T4ligase (Sambrook, Fritsch et al. 1990) to fdrVabpCARRIER (see FIG. 11)precut with Not1 and Apa1. The ligated product, designatedfdrVabPEPpCARRIER is isolated with GeneClean and electroporated into E.coli. Transformants are grown, tittered and frozen stocks are made. Asufficient number of colonies are picked and sequenced to confirm thepresence of the random pep8 library. The bacteria, designatedGHRT(x-y).rVab-PEP.lib.bact are then grown and phage are induced forexpression with helper phage so that the GHRT(x-y) rVab-pep constructsare displayed on the phage surface attached to gpIII. (see FIG. 20).

With the amino acids of the octapeptide being random at each position,there are greater than 10¹⁰ peptide combinations for each library.Accordingly, with less than about 100 GHR.TS+rVab in each group thecombinatorial rVab-pep.lib number is less than 10¹² and is thereforeaccommodated in a normal phage lysate. If the number of GHR.TS+ rVab isgreater than about 100, the random octapeptide library is expressedalone as a fusion protein fused to the gpIII on the surface of fd phagevia the same linker ([GGGGS]2) and the octapeptides which recognize GHRsurfaces are isolated first by panning over matrix attached GHRcomplexes. Those phage which stick to the matrix, are isolated,amplified and the oligonucleotide sublibrary encoding the pep8octapeptides which bind to GHR are excised and amplified with primerscontaining a leader restriction site (in the BCK primer) and ApaL1 (inthe FWD primer). This smaller pep8 oligonucleotide sublibrary, which isT+ (pepT+), is then ligated into the grouped GHR.TS⁺ rVab.lib precut atthe rs1 site in the VL Lgp111 leader secuence and at Apa 1 (See FIG.11D) to produce a GHR.TS+rVab-pep(T+) library. In such cases the membersof this combinatorial library, less than 10¹⁴, are grown, the phageinduced and the library of surface attached GHR.TS+rVab-pep(T+)harvested and tittered.

Step 2b: Identification of Active Bivalent rVab-Pep Members

2b) Isolate GHR(x-y) T+.rVab-pep members which actively (A+) dimerizethe receptor as does GH. Label these GHR(x-y)TA+rVab-pep.

The bivalent; rVab-PEP, are expressed as a phage displayed library andare panned for combinatorial members which actively dimerize GHR. Thepositives are labeled GHR.rVabT(x-y)SA+-pepT1+.lib. In this step,activation is recognized by the occurrence of one or more of thefollowing observable events: 1) dimerization of two GHR T1 subunits; 2)dimerization of two T1 subunits which allow fluorescence transferbetween the same or different modified amino acids in the two subunitsas described by Cunningham (Cunningham, Ultsch et al. 1991); 3) dimmerformation which generates an antibody recognized epitope which containsamino acids from two T1 subunits which occur only in activated dimericT1² structures (Taga, Narazaki et al. 1992); 3) GHR-GH-GHR-matrixcomplexes which are dissociated by wild type hGH, or only a mutant hGHwith only site I or site II binding capability (Cunningham, Ultsch etal. 1991); or 4) antibody recognizable phosphorylation of one of thereceptor subunits associated with active receptor dimerization. In thelater case, incubation of GHR.rVabT(x-y)S+pep.lib with ATP and PKC iscarried out before panning and the ATP and PKC is present during thepanning procedures. It is also possible to monitor for in vitro activedimerization by the co-presence of some third GHR associated protein inthe active complex (Taga, Narazaki et al. 1992).

2c) Confirm activity by testing for activation of a cell associated CGHR. Those GHR.TSA+rVab-pepT which appear active in vitro, are tested inan intact cell assay system such as GH induced growth of myeloidleukemia cell line FDCP1 expressing hybrid extracellular domainGHR-intracellular granulocyte colony-stimulating factor receptor (GCSFR)(Fuh, Cunningham et al. 1992) or IM-9 cells (Silva, Weber et al. 1993)to confirm the agonist nature of the rVab-pep complex.

3. Identification of Active GH-rVab Pairs for use as Reporters

Step 3a. Expression of Soluble rVabs

3a) Identify from among the members of different A+ rVabA+-pep groups,those which have a rVab which by itself competes with the peptide memberof the same or different rVabA+-pep group. This is accomplished bycarrying out competition binding assays designed to identify those rVabsand peptides which compete with each other for binding to the GHR. Thepeptide portion of an active rVab-pep is separately expressed withoutthe corresponding rVab to perform these binding assays. By this processrVabs which can mimic and replace the pep8 portion of an active rVab-pepmember are identified. The rVab of a first A+rVab-pep member and therVab of a second A+rVab-pep member which competes with the peptideportion of the first member, are designated an active pair of GH-rVabs.

Specifically, after confirmation of activation is obtained, the activerVab-pep are modified by appropriate digestion of the construct to allowexpression of soluble rVab without any linkage to phage coat proteingpIII and to the octapeptic e as well. Such simplified entities arelabeled rVabTS+A*. To prepare the modified constructs allowing forexpression of free soluble rVab, DNA from rVab-pep is obtained, digestedwith Apa1 and Not1 and isolated. One μg of the isolated DNA is thenligated with 5 μg pEXPRESSIONrVab DNA precut with ApaL1 and Not1 byincubation with T4 ligagse. The ligated products are isolated byGeneClean II and electroporated into E. coli and transformants obtainedand confirmed by diagnostic PCR and sequencing. Frozen stocks areprepared. These stocks are denoted GHR.rVabTS+A* and not A+ because bythemselves they cannot activate the GHR but are members of active pairs(i.e., rVabs and pep8s) which do activate the receptor. Expression ofthe octapeptide member of the active rVab-pep is carried out by excisionand ligation of the oliognucleotide portions encoding the pep8 andtransfer to expression vectors in which the pep8 is expressed as asoluble extracellular entity fused with a easily purifiable taggedcarrier protein (using a variety of commercially available expressionvectors) or attached via GGGGS linker to gpIII coat protein anddisplayed as a phage surface entity. These entities are labelled pep8A*and are used as described below to identify rVab for the other portionof the GHR active surface utilized by the active rVab-pep entity.

3b) rVab and pep8 members of active pairs are grouped according tocommon GHR surfaces recognized (as described above).

4. Preparation of GH-rVab-Reporters

Convert a rVab representative of at least one active pair of GH-rVabsinto a GH.rVab-Reporter.

The CH domain of the heavy chain of the rVab is labelled (as describedin Example 2) and the labelled entity, designated GH.rVab-REPORTER, isused to establish saturation and competition binding assays as describedin Example 2.

The isolated and expressed separated pep8 members from active rVabA+-pepconstructs are used in standard binding competition assays to identify(see FIG. 11) those GHRrVabT+ which bind to the same GHR domain as thepep8 entities. Those which compete are designated as the second memberof the active pair of rVab for the two active GHR surfaces required forreceptor activation. This second member is then converted to arVab-Reporter (see above). The rVab member of the rVabA+-pep constructfrom which the pep8 was obtained is the second member of the activepair.

Step 5: SOMER SCREENING

Establish binding assays with each member of an active pair ofGH.rVab-REPORTERs for a pair of SOMERS, each capable of binding to atleast one of the two domains of an active pair of receptor surfacesinvolved in active GHR dimerization.

The GH.rVab-REPORTER is used under standardized and automated bindingassay conditions to identify SOMERs within a chemical data base (i.e.,CHEMFILE) which will compete at an active* (A*) surface on the T1subunit of the GH receptor. These SOMERs are designated SOMER-T1. In aparallel fashion, using the other rVab-Reporter member of the activerVab pair (as defined above) SOMERs are isolated for the second activesurface on GHR required for its activation (FIGS. 21 and 22). The SOMERswhich recognize the second site are designated SOMER-T1.

Identification of specific interaction with site I (i.e., T1) or site II(i.e., T1') of huGHR is made in binding assays measuring the ability ofthese entities to compete with mutant 125I-GH which can only bind tosite I or II as described (Cunningham, Ultsch et al. 1991).

Step 6: DISOMER Preparation and Identification of Drug Leads

In the last step of this process, SOMER-T1 and SOMER-T1' are covalentlycombined to create a bivalent SOMER (i.e., a DISOMER) which canrecognize the two sites of the active surface pair, i.e., the T1 and T1'receptor dimmer subunit active surfaces. This DISOMER can activelydimerize the GH receptor subunits as does the native hormone.Confirmation of DISOMER GH activity is obtained in standardradioreceptor binding assays (competitive with intact labelled (GH) forGHR binding and standard activity assays (in vitro and/or GHR cellularactivation systems). Additional assay systems for active hormonereceptor subunit oligomerizations in which a free excellularreceptor:hormone complex associates with other membrane proteins inintact cells to form active oligomeric complexes which direct auto-, andsubstrate phosphorylation, and other down stream activation responses(Taga, Narazaki et al. 1992).

Steps 1-4 of the process, which find active surface landscapes involvedin active dimerization of two TI subunits of GHR are outlined in FIGS.20, 21 and 22. FIG. 20 is a flow diagram for creation of rVab-pep.libraries and isolation of rVab-peptides for the two active GHRsurfaces. In the example presented here of oligomeric receptor targets,there is only one type of subunit (T1) in the active GHR dimer complex,and therefore subunit T2=T1. FIGS. 21, 22 illustrate GHT1- andGHT1'-SOMER and GH-DISOMER (i.e., GHT1-GHT1') identification.

EXAMPLE 4

Example 4 is a variation of Example 3 which recognizes the fact thatmany hormonal receptors are comprised of different receptor subunits.Often at least two or three subunits which may all be different fromeach other are required for activity. In these cases, hormone inducedreceptor oligomerization associated with receptor activation, requiresinteraction of the hormone with at least three active surfaces, eachbeing on a different receptor subunit. Examples of heterodimeric(alpha/beta, or alpha/gamma) receptors include the group of interleukin(IL) IL3, IL4, IL5, IL7, IL9 receptors and the GMCSF receptor, and thegroup of growth factor FGF, PDGF, CSF and NGF receptors, while anexample of a heterotrimeric receptors (alpha, beta and gamma) is the IL2receptor (see reviews Pierce, 1989; Boulay, 1993; Cosman, 1993;Kishimoto, 1994; Kaushansky, 1993; Kondo, 1994; Noguchi, 1993; Russell,1993 and Bamborough, 1994).

The use of rVab to identify active surfaces involving two or more site'sdistributed on multiple subunits involves certain adaptions from theprocess used when activation requires only one site. First, with theheterooligomeric receptors, a different rVabT(x)S+ lib is identified foreach subunit (x) using the soluble receptor subunits as initial targets(e.g. Tavernier, 1991), as Second, that for trimeric receptors tworandom peptide 8 libraries are attached to each rVabT(x)S+ library.Third, where the rVab is T+ for the alpha receptor subunit (i.e.,rVabT+S), the other two members of the active trio (i.e., those bindingto each of the other two subunit surfaces necessary for active receptortrimerization), designated rVabT+ and rVabT+, are identified as thosewhich compete for binding with one of the two octapeptide members of anactive rVabTSA+-pep². For such trimeric receptors, the individualrVHCH.lib and rVLCL.lib made in Example 1 are combined into differentfdRECEIVERs and pUC19PROVIDERs as detailed in FIG. 13.

In this application, rVHCH.lib is placed into a fdRECEIVER which allowsexpression of rVHCH fused to gpIII coat protein and with, or without,peptide (preferably 8 amino acids) attached to its aminoterminus. TherVLCL.lib is placed into a pUCPROVIDER which allows for expression ofrVLCL as soluble entities with, or without, peptide, preferably 8 aminoacids, attached to its CL domain. After in vivo Cre-Lox -recombinationof these two libraries, as detailed in Example 1, (see also. FIG. 13)the product rVab.lib is cloned as a single fdDNA designatedfdrVabPEPCARRIER. rVab members which bind to each of the receptorsubunits (i.e., Tx+rVab) are then isolated and grouped as described inExample 3. Subsequent addition of one or two random octapeptidelibraries (Pep 8^(n)), which in some cases have been prescreened andselected for binding to an identified receptor subunit is accomplishedvia PCR. As described above and in FIG. 13, oligonucleotides encodingthe peptides are added to the DNA encoding the rVab library using FWDprimer CLLNKPEPFWD (Asc1-(NNN)8(GGGGS)3CLL208-216) and VHLNKPEPBCK(rsPELB-(NNN)8(GGGGS) VH1-8) together or in combination with primershaving no Pep8 or linker- appending sequences. Use of one of theseprimers with a primer devoid of a Pep8 library could be used to generatea rVab with one attached pep8 (i.e., rVab-Pep 8¹) as described above inExample 2 with the single Pep 8 library appended through linker to theeither the aminoterminus of the rVHCH1 member or the carboxyterminus ofthe rVLCL member (FIG. 13). According to this process, each attachedpeptide and the rVab portion of the rVab-PEP² each bind to a specifictarget site. Binding to all three sites is required for activity of thereceptor. Therefore, the trimeric rVab-PEP² unit defines three bindingdomains: one defined by the rVab portion ((T(x)), and one each by eachof the pep8 (i.e., pep8¹ and pep8 ²) present in the construct.

Isolation of active rVab-Pep² members utilizes enrichment cycles inwhich all three receptor units are complexed together in active trimericstructures. Such structures, complexed with their phage expressedrVab-Pep² entities, are enriched by use of matrix-bound active subunits,antibodies to each of the three units, antibodies to modifications ofreceptor units which occur upon active oligomerization, such asphosphorylation or association with additional non-receptor membranecomponents (Argetsinger, Campbell et al. 1993; Silvennoinen, Witthuhn etal. 1993; and Witthuhn, 1993). Confirmation of agonist and antagonistactivity is done using standard hormone: receptor binding assays toestablish competitive binding of hormone to its receptor (Kitamura, Satoet al. 1991; Imler and Zurawski 1992; Pietzho, Zohlnhofer et al. 1993)and cellular receptor dependent activity assays measuring growth, DNAsynthesis, protein phosphorylation etc. (Yokota, Otsuki et al. 1986;Pierce, Ruggiero et al. 1988; Solari et al. 1989; Anklesaria, Teixido etal. 1990; Heidaran, Pierce et al. 1990; Pierce, Di et al. 1990;Heidaran, Pierce et al. 1991; Keegan, Pierce et al. 1991; Murakami,Narazaki et al. 1991; Kruse, Tony, et al. 1992; Otani, Siegel et al.1992; Taga, Narazaki et al. 1992; and Wang, Ogorochi et al. 1992).

For a given rVab-Pep8² we identify rVabA*s which are contained in otherrVab-Pep8² A+ which bind to each of the target sites bound by thepeptides of the original active rVab-Pep8² (trimer rVabA* unit),following the same process outlined in Example 3. Using this process,members of the trimeric unit are identified as a) any rVabT+ fromanother active construct (i.e., rVabTSA+-Pep8²) which competes with oneof the two PEP lib on the original active rVabTSA+-PEP², b) with anyrVabT+ from a third active construct (i.e., rVabTSA+-Pep8²) whichcompetes with the other PEP on the original active rVabTSA+-PEP² and c)the rVabTS+ of the original active rVabTS+-PEP². Competition for bindingto GHR is determined by assaying for competition of PEP8 units expressedeither attached to gpIII coat protein and presented as phage displayedentities or as soluble fusion proteins with labelled rVabTx+-Reporterswhich are made as described above in example 2 and 3. Afteridentification of all three of the active trimer members, each rVabmember of the active timeric unit is then cloned minus its Pep8librarymember(s), expressed, isolated and converted to a rVab-REPORTER, asdetailed in Example 1, and used to establish competitive binding assayswhich then find competing SOMERs (i.e., Somer-T₁, T₂ or T₃). In thefinal stage covalent linking of the three Somer-Ts) is done so as toconstruct the active multimer, in this case a TRISOMER (i.e., T₁ -T₂-T₃), substitute for the native hormone. In these systems, an additionalreceptor activation assay system is available for heterooligomericreceptor activation which monitors the induction of identifiableholoreceptor induced cellular responses by preformed soluble complexesof hormone and one of the receptor subunits in response to the bindingof these complexes to intact cells expressing the other subunit(s) ofthe active receptor complex and formation of active holoreceptorcomplexes (Taga, Hibi et al. 1989).

In these systems, an additional receptor activation assay system may beused to confirm heterooligomeric receptor activation. Such systemsmonitor the induction of identifiable cellular responses induced by thecombination of preformed soluble complexes comprising hormone and one ofthe receptor subunits and intact cells expressing the other subunit(s)of the active receptor complex and the subsequent formation of activecomplete holoreceptor complexes (Taga, Hibi et al. 1989).

The following Table lists exemplary ligands and heterooligomericreceptor systems for which this invention provides a means foridentifying their pharmacologic target sites as well as SOMERS orDISOMERS.

    ______________________________________                                        Interleukin1                                                                            Immune System Supression/                                                                          Agonist/                                                 Stimulation          Antagonist                                     1L2-7, 9-11                                                                             Immune System Supression/                                                                          Agonist/                                                 Stimulation          Antagonist                                     Insulin Like                                                                            Neoplasias           Antagonist                                     Growth Factors:                                                                         Erythropoiesis       Agonist                                                                       (synergistic                                                                  w Epo)                                                   Granulopoiesis       Agonist                                                                       (synergistic                                                                  w GMSCF)                                       TGFbetas  Wound Healing (Matrix proteins)                                                                    Agonist                                                  Inflammation         Antagonist                                               Carciogenesis        Antagonist                                               AutoImmune Disease   Antagonist                                     GCSF      Chemotherapy         Agonist                                                  Bone Marrow Transplation                                                                           Agonist                                        CSF       Bone Marrow Failure Syndromes                                                                      Agonist                                                  (re: radiation/chemotherapy)                                                  Inflammatory         Antagonist                                               Neoplasms (acute myeloid leukemia)                                                                 Antagonist                                     Erythropoietin                                                                          Hematopoiesis (anemias)                                                                            Agonist                                        GMCSF     Immune Suppression/Stimulation                                                                     Agonist/                                                                      Antagonist                                     PDGF      Wound Repair         Agonist                                                  Angiogenesis         Antagonist                                               Vasoconstriction     Antagonist                                               Atherosclerosis      Antagonist                                               Neoplasms            Antagonist                                               Pulmonary Fibrosis   Antagonist                                               Inflammatory Joint Diseases                                                                        Antagonist                                     EGF       Wound Repair         Agonist                                                  Neoplasms            Antagonist                                     FGF       Neoplasms            Antagonist                                               Wound Repair         Agonist                                                  Angiogenesis (Capillary Blood                                                                      Antagonist                                               Vessels)                                                            NGF       AntiNeurodegenerative Diseases                                                                     Agonist                                                  (Acute/Chronic); (Peripheral/Central)                               Small Organic                                                                           Neurotransmitters    Agonist/                                       Molecules i.e. Cholinomimetics (ACh @                                                                        Antagonist                                               mReceptor 1-5)                                                                Transporter/Channel Regulators                                                                     Agonist/                                                                      Antagonist                                     ______________________________________                                    

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While we have hereinbefore described a number of embodiments of thisinvention, it is apparent that the basic constructions can be altered toprovide other embodiments which utilize the methods and compositions ofthis invention. Therefore, it will be appreciated that the scope of thisinvention is defined by the claims appended hereto rather than by thespecific embodiments which have been presented hereinbefore by way ofexample.

I claim:
 1. A method of identifying a potential drug candidate capable of binding to at least one site of a biologically active target, which site is capable of conferring a biological response, and wherein said potential drug candidate exhibits either agonist or antagonist activity at the target, the method comprising:a) providing at least one detectable reporter of binding of said potential drug candidate to the biologically active target, wherein said reporter possesses agonist or antagonist activity at said target and wherein said reporter is selected from a recombinant library of antibodies comprising at least one variable region; b) screening potential drug candidates by assaying for the ability of the potential drug candidates to compete with the reporter for binding to the target; and c) selecting potential drug candidates possessing agonist or antagonist activity, which compete with the reporter for binding to the target.
 2. The method according to claim 1 wherein each antibody member (rVab) of the antibody library comprises at least one variable region selected from the group consisting of VH and VL regions, and optionally comprises a constant domain attached by its amino terminus to the variable region.
 3. The method according to claim 2 wherein the rVab unit is displayed on the surface of a carrier.
 4. The method according to claim 2 wherein the rVab unit is soluble.
 5. The method according to claim 3 wherein the carrier is a bacteria.
 6. The method according to claim 3 wherein the carrier is a bacteriophage.
 7. The method according to claim 2 wherein a parental VL region comprising at least one CDR is used to derive the VL region of the rVab by deleting, inserting or substituting at least one amino acid within at least one CDR.
 8. The method according to claim 2 wherein a parental VH region comprising at least one CDR is used to derive the VH region of the rVab by deleting, inserting or substituting at least one amino acid within at least one CDR.
 9. The method according to claim 2 wherein parental VL and VH regions comprising at least one CDR, are used to derive a pair of VL and VH regions of a rVab by deleting, inserting or substituting at least one amino acid within at least one CDR of each variable region.
 10. The method according to any one of claims 7, 8 or 9 wherein the crystal structure of the parental V regions used to derive rVab are known.
 11. The method according to claim 9 wherein the crystal structure of the parental VH and VL pair used to derive the rVab is known.
 12. The method according to claim 2 wherein at least one of the parental V regions used to derive rVab is unmodified.
 13. The method according to claim 2 wherein the crystal structure of the rVab is determined after isolation as a rVab which binds to a biologically active site on the target.
 14. The method according to claim 2 wherein at least two V regions are modified by deleting, inserting or substituting at least one amino acid in at least one CDR after isolation as rVab which binds to a biologically active site on the target.
 15. The method according to claim 1 wherein the target is a polypeptide, protein, nucleic acid, oligosaccharide, carbohydrate or lipid.
 16. The method according to claim 1 wherein target activation by the reporter is determined by assaying a biochemical response at the target which biochemical response occurs subsequent to binding of the reporter to the target and is a signal of target activation.
 17. The method according to claim 16 wherein the biochemical response is detectable as a change in a protein or polypeptide characteristic.
 18. The method according to claim 16 wherein the biochemical response is associated with an organometallic moiety, a metal or other nonprotein.
 19. The method according to claim 16 wherein the biochemical response comprises a detectable free radical, fluorescent or chemiluminsecent group, radioactive isotope or involves oligomerization.
 20. The method according to claim 16 wherein the biochemical response is phosphorylation and the signal is a change in the phosphorylation state of the target.
 21. The method according to claim 17 wherein the signal protein is a G protein and the signal is a change in either the presence of a G protein regulatory agent or the binding of rVab due to the presence of a G protein regulatory agent.
 22. The method according to claim 16 wherein the signal is a change in the binding affinity of rVab to its binding site.
 23. The method according to claim 2 wherein the recombinant antibody comprises a single polypeptide chain comprising a VH region and a VL region which together form a binding site.
 24. The method according to claim 1 wherein the target is a eukaryotic target.
 25. The method according to claim 1, wherein the recombinant antibody library comprises at least 10⁹ members.
 26. The method according to claim 1, wherein the recombinant antibody library comprises at least about 10¹² members.
 27. The method according to claim 9, wherein the parental VL and parental VH each comprise randomized amino acids at least one position of at least one CDR.
 28. The method according to claim 9, wherein the parental VL region comprises a CDR1, a CDR2 and a CDR3.
 29. The method according to claim 28, wherein at least one CDR comprises a multiplicity of canonical structures.
 30. The method according to claim 29, wherein the CDR1 comprises five different canonical structures, CDR2 comprises one canonical structure, and CDR3 comprises six different canonical structures.
 31. The method according to claim 9, wherein the parental V_(H) region comprises a CDR1, a CDR2 and a CDR
 3. 32. The method according to claim 31, wherein at least one CDR comprises a multiplicity of canonical structures.
 33. The method according to claim 32, wherein CDR1 comprises three different canonical structures, CDR2 comprises four different canonical structures, and CDR3 is devoid of canonical structures. 